M.Sc. THESIS (Biomedical Engineering) : EFFECTS OF LOW LEVEL LASER THERAPY ON HUMAN BONE REGENERATION.

1. .
Gono- bishwabidyalay (Gono-University),Mirzanagar, Savar, Dhaka-1344,Bangladesh.April
2010.
1
Dept.of Medical R adiation P hysics,Kreiskrankenhaus Gummersbach, Teaching Hosp ital of the University of Cologne,
51643 Gummersbach, Germany.
2
Dept. of Medical Physics and Biomedical Engineering, Gono-Bishwabidyalay ( Gono University), Nayarhat, Savar,
Dhaka- 1344, Bangladesh.
3
Department of Orthopedic and Traumatology, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207,
Bangladesh.
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2.  Declaration
I declare that I am the sole author of this thesis and that the work presented
here has not previously been submitted as an exercise for a degree or other
qualification at any university.
It consists entirely of my own work, except where references indicate
otherwise.
Dr. Md. Nazrul Islam. 10th April/2010.
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3. Dedication
To my parents, Mr. Alauddin Sikder & Ms. Munira Begum- for fostering and
encouraging my interest in science
&
Mother in-law, late Ms. Anowara Begum and my wife Ms. Habiba Islam
Happy for their tremendous and unbelievable mental support during my post-
graduate period.
Sponsored by-
This research project is jointly sponsored by-
Lab-Nucleon & Acme Laboratories Ltd., Dhaka, Bangladesh.
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4. I wish to express my deepest gratitude and thanks to my guide and thesis supervisor Professor Golam Abu Zakaria, for his
kind directions, inspiring guidance, and invaluable discussion throughout the course. Without his patience and
encouragement, this work would never be fulfilled. I also express my heartfelt respect to my co-guides Prof. F. H. Sirazee ,
ex. head of the department, and Associate professor, Dr. P C Debenarh, Department of Orthopedic & Traumatology,
Shaheed Suhrawardy Medical College Hospital. Dhaka-1207, Prof. Nurul Islam dept. of Medical Physics and
Biomedical Engineering, and Md. Delwar Hossain, registrar,Gono Bishwabidyalay (Gono University) Nayarhat,
Savar, Dhaka for heartiest co-operation and advice throughout the whole research period.
I would also like to express my profoundest gratitude to my thesis advisory board / working team members, Associate Prof.
Dr Sheikh Abbasuddin Ahmed, assistant Prof Dr. Kazi Shamimuzzaman, Dr. Zia Uddin,consultant, Dr. Subir Hossain
Shuvro, assistant registrar, Dr. Abdul Hannan of Orthopedic & Tramatology Department, assistant Prof Dr. Quamrul Akter
Sanju of Surgry department, Shaheed Suhrawardy Medical College Hospital and Dr. Sayed Shaheedul Islam Assistant
professor, NITOR, Dhaka.
Special thanks are due to Prof. Khadiza Begum, ex-director, Prof. A. K.M Mujibur Raman, director, Dr. Mir Mahamuda
Khanam, assistant director of SsMCH, Prof. S. M. Idris Ali, ex. head of the department and vice-principal,SSMC and Prof.
Abdul kader Khan, ex-principal and head of the department, surgery, SSMC/ SsMCH, associate professor, Dr. Mostaq
Hossain Tuhin of Surgery department, assistant. Prof. Asraf Uddin, head o f the department ,Radiology & Imaging
department of SsMCH, and to all my friends and well-wishers, specially to Mr. Sinha Abu Khalid CEO, LabNucleon, Md.
Masud Rana, medical Physicist, National Cancer Institute & Hospital, Md. Anwarul Islam, medical physicist, Squrae Hospital
& Mr. Kumaresh Chandra Pal, medical physicist of Gono Biswabidyalay, Dhaka-1344, and Md Shakilur Rahman, senior
scientific officer of Bangladesh Atomic Energy Commission for their kind and nice co-operation throughout the course of this
work.
My deepest admiration and sincerest love to and my laser machine technician Mr. Mamun, laser operator Ms. Jannant &
Ms. Chewty of LabNucleon, and Md. Abdul Aziz, Mr. Polash, Mr. Abul- kasem, Ms. Fatema, Ms Farida, Mr. Malek of
Orthopedic and Traumatology Department, and Kazi Murad Hossain of Shaheed Suhrawardy Medical Hospital, Dhaka-
1207, Bangladesh for their continuous efforts to successfully complete this project.
Finally, I owe much and pay my heartiest thanks, especially to those who rendered their hind assistance during my study
period. Words cannot be expressed my feeling of love, I am deeply indebted to my wife Habiba Islam, my loving sons-
Sayem Islam labib & Talaat Islam Syiam for all their support and understanding. In the whole course of this work, they gave
me a sweet working atmosphere, which I can‘t find words to express.
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5. BY-
D R. M D NAZRUL ISLAM
Laser (Semiconductor diode, Ga-Al-As, 830nm) is effective in human bone regeneration,
i.e. it enhances bone fracture healing.
Tissue healing is a complex process that involves both local and systemic responses, and the healing process of bone is
much slower than that of soft tissues which is a great challenge of medical science. The use of Laser Therapy (LLLT) for
wound healing has been shown to be effective in modulating both local and systemic response by enhancing- cellular &
mitochondrial ion exchange, bone mineralization, nitric oxide formation, lymphatic circulation, osteoblast proliferation, eff ects
on osteoblast gene expression, osteoclast inhibition (prevents bone mineral resorption) and by bone engraftment on
synthetic materials.
40 (Twenty in laser & Twenty in control group) otherwise healthy men and women with, closed appendicular bone fracture
(Radius/ ulna, or Femur / Tibial shaft /Clavicle / Meta carpal /Meta-tarsal) was enrolled for fracture management by laser
therapy adjunctive to regular management, and was assed by clinical and radiological findings (X-ray)/at 2nd , 3rd, 4th and
6th week post fracture: assessment included fracture line/margins, fracture gap, external callus appearance, callus-to-cortex
ratio, bridging, and radiologic union as well as clinical assessment of the fracture- compliance of patient, and onwards
follow-up of patients, in comparison to controlled group.
Early significant bone regeneration /callus formation achieved by early application of Low Level laser therapy (Ga-Al-As, 830
nm) on human fractured long (appendicular) bone.
Treatment with 830 nm diode laser has substantially reduced the fracture healing time as well as improved the
quality/quantity of callus formation of the patient, thus enhancing fracture healing. Laser biostimulative effects on bone could
be a new dimension for bone regeneration which significantly reduce healing period, lessen cost of treatment, and enhance
patient compliance in medical science.
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6. Introduction, Background and literature review
1.1 Introduction
1.2 Background
1.3 Literature review
Cell & Bone
2.1 Cell
2.2 Bone
2.3 Bone Fracture
2.4 Healing of fracture
Laser & Laser System
3.1 Laser
3.2 Laser principle
3.3 Components of a laser system
3.4 Laser Machine
3.5 Measurement of Laser Energy
Biophysical Aspects, Laser-Tissue Interaction, Mechanisms and Bone Regeneration.
4.1 Biophysical Aspects & light transport theory
4.2 Laser - Tissue Interaction
4.3 The Mechanisms of Low Level Laser Therapy
4.4: Effects of Laser on Biological Cell/Tissue healing
Laser on hard tissue & Bone stimulation/ Regeneration
4.5 Medical application of Low Level Laser
Materials & Methods
5.1 Materials
5.2 Methods
Observation & Result
Discussion
Conclusion
References
Appendices-
10.1 Figure & Table list
10.2 Laser Books & Articles
10.3 Datasheet.
10.4 Publications & Presentation:
10.5 Biography & pictures
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7. Chapter-1 Page- 7- 12
1.1 Introduction
1.2 Background
1.3 Literature review
Optimizing the results of fracture treatment requires a holistic view of both patients and treatment. The nature of the patient
determines the priority targets for outcome, which differ widely between the elderly and the young, and between the victims
of high and low energy trauma. The efficacy of treatment depends on the overall process of care and rehabilitation as well
as the strategy adopted to achieve bone healing.
The rational basis for fracture treatment is the interaction between three elements, (I) the cell biology of bone regeneration,
(ii) the revascularization of devitalized bone and soft tissue adjacent to the fracture; and (iii) the mechanical environment of
the fracture. The development of systems for early fracture stabilization has been an advance. However, narrow thinking
centered only on the restoration of mechanical integrity leads to poor strategy - the aim is to optimize the environment for
bone healing. Future advances may come from the adjuvant use of molecular stimuli to bone regeneration.
Restoring function to a patient who has had a fracture requires the physician/ surgeon to handle a heady mix of mechanical
and biological issues. In real life, it also requires considerable input of time into practice organization, given the large
numbers of patients and the almost universal inadequacy of resource, if each individual patient is to receive timely and
appropriate intervention.
There is a perception, not least among fracture surgeons themselves, that the mechanical issues have been over-
emphasized in the past. The bonesetter's art consisted basically of- providing- anatomical realignment and external support
for as long as nature then took to restore internal structural competence by bone healing. This was slow and unkind to soft
tissues, particularly neighboring joints, so the development of materials, bio-mechanical understanding and surgical
technique launched a swing towards invasive interventions aimed at immediate restoration of internal structural integrity.
The principles of AO treatment, drummed into a generation of orthopedic trainees, were anatomical open reduction, rigid
internal fixation and early rehabilitation of soft tissues without- external splint. But the scale of invasion required to achieve
these aims brought a steady trickle of serious problems - most notably infected non-unions, sometimes in cases which
surgeons knew they could safely have treated by simpler methods.
Furthermore, there was increasing realization that the abolition of inter-fragmentary motion implied a commitment to primary
cortical union as the only route for healing and a closure of the natural routes of callus formation. From various directions,
less invasive alternatives were developed: functional bracing, external fixation (including the remarkable Ilizarov circular
fixator developed in the USSR, which evades the bone only with fine wires) and closed intra-medullary nailing.
Now the science is taking another step, further in the direction from mechanics to biology. If the mechanical environment
influences bone regeneration and hence fracture healing, how, at a cellular- level, does it do so? - What molecular signals-
produce the response? If we know the signals, can we deliver them in the form of recombinant growth factors and hurry the-
cellular response down the right path?
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8. The evolution has been first to use nature, then to ignore her, then to remember her, and now to outdo her.Optimal fracture
treatment requires the following: (I) a definition of what optimal treatment means and a way of measuring the extent to which
it is achieved; (ii) a review of what we know about- the natural healing process that we want to harness or improve upon;
and (iii) analysis of how to apply the above to clinical practice.
During the last decade, it was discovered that low-power laser irradiation has stimulatory effects on bone tissue, in the
microscopic (cell proliferation [1-5] and gene expression [6]) and macroscopic [1, 2, 4, 12, 13-20] biological systems.
In order to understand the effects of laser therapy, its mechanism of action in the cell needs to be established. Many
explanations have been proposed {7-11]. Studies have shown that porphyrins and cytochromes, natural photoacceptors
located in the cell, are the main contributors to laser-tissue interaction [7-11]. Porphyrins and cytochromes absorb the light into
the cell, resulting in the production of singlet 1O2. The singlet oxygen then stimulates the redox activity in the mitochondria,
enhances chemiosmosis, DNA production and calcium-ion influx into the cytoplasm, thereby causing mitosis and cell
proliferation.
The purpose of this review is to analyze the effects of low power laser irradiation on bone cells and bone fracture repair,
examine what has been done so far, and propose areas warranting further exploration.
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9. Bone and fracture healing is an important homeostatic process that depends on specialized cell activation and bone
immobility during injury repair [1, 2]. Fracture reduction and fixation are a prerequisite to healing but a variety of additional
factors such as age, nutrition, and medical co-morbidities can mediate the healing process [3, 4]..Different methods have been
investigated in attempts to accelerate the bone-healing process. Most studies have concentrated on drugs, fixation methods
or surgical techniques; however, there is a potential role for adjunctive modalities that affect the bone-healing process.
Laser is an acronym for ―Light Amplification by stimulated Emission of Radiation ‖ [5]. The first laser was demonstrated in
1960 and since then it has been used for surgery, diagnostics, and therapeutic medical applications [6]. The physiological
effects of low level lasers occur at the cellular level [7, 8], and can stimulate or inhibit biochemical and physiological
proliferation activities by altering intercellular communication [9]. Early work on physical agents as mediators of bone healing
was performed by Yasuda, Noguchi and Sata who studied the electrical stimulation effects on bone healing in the mid
1950s [1, 10]. In subsequent years, others repeated this work in humans [1, 11] and a variety of physical agents have been
investigated as potential mediators of bone healing [12, 23, 14, 15, and 16]. With increasing availability of lasers in the early 1970s,
the potential to investigate its use as a modality to affect the healing of different connective tissues became possible [17, 18, and
19]. In 1971, a short report by Chekurov stated that laser is an effective modality in bone healing acceleration [19].
Subsequently, other researchers studied bone healing after laser irradiation using histological, histochemical, and
radiographic measures [18, 19, 20, 21, 22, 23, and 24]. These studies have demonstrated mixed results where some observed an
acceleration of fracture healing [19, 21, 22, 23, 24], while others reported delayed fracture healing after low-level laser irradiation
[20, 25].
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10. As far as is known, the first attempt at treating bone fracture with infrared light was reported by Shugaharov and Voronkov.
In 1974 they used low level laser radiation (infrared wavelengths) on fracture sites observing intramedullary osteosynthesis.1
Gatev studied the effect of stimulating repair of fractures with He-Ne laser. The majority of patients had fractures of the distal
radius treated with a plaster cast. On the 5th to 8 th day after injury a hole was cut out of the cast over the fracture site and
laser radiation applied at 632 nm, 2 mW/ cm2. Evaluations were made based on radiographic evidence and clinical
assessment. Results showed statistically significant differences [p<0.001] from the control group in favor of light treated
fractures.2
A 1990 case study looked at a non-union long bone fracture refractive to treatment over a period of 8 months. A 24 year-old
patient was treated conservatively for displaced fracture of the diaphysis of both bones of the right forearm. When secondary
displacement occurred the fracture was operated on with use of a compression plate for the radius and a single Rush Rod
for the ulna. Eight months after the injury the radiological and clinical examination showed signs of delayed union of both
fractures. A diode laser emitting 890 nm wavelengths near infrared light with average output of 3 mW and an energy
deposition of 1.8 Joules/cm2 was applied 3 times per week. After 4 weeks of treatment the signs of callus formation
appeared.
After another 5 weeks the radiogram showed complete remodeling of the ulnar bone and union in the radius. No side effects
were observed.
Figure1.3.1: Before & after laser therapy.
A 15 year old male athlete presented with an avulsion fracture with involvement of the inferior aspect of Anterior Superior
Iliac Spine. ASIS injury was non-weight bearing. Patient was taking 3200 mg ibuprofen daily.
Normal prognosis is 4-6 weeks non weight bearing followed by 6 weeks of rehab and additional 10 weeks before return to
sport (running). Protocol followed for this case: initiated daily infrared light treatments, 890 nm, 20 Joules/cm2, 20 min
treatments. Rehab begun on third visit. Discontinued ibuprofen after third treatment. Discharged from treatments after 24
visits and orthopedist released patient at 100% to return to running. Total time reduced from 22 weeks (normal prognosis) to
5 weeks.4
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11. An 18 year old high school athlete presented with a non union tibial fracture. The patient had previously fractured the same
site, taking 15 months to heal. Re-fracture was fixed with a compression plate. After 2 years the patient still showed edema
and pain with radiographic evidence on non union.
Once daily treatments (5 days per week) were initiated with infrared light, 890 nm, 20 Joules/cm2, 20 min per treatment.
After 44 days radiographic analysis showed pannus formation over the set screws. After 86 days radiograph showed
complete fracture healing.24 Note pannus formation over screws. 5
A patient presented with a non union 5th metatarsal fracture of the left foot. The patient was treated conservatively with
immobilization and non weight bearing. After 3 months no progress was evident from radiographic and clinical assessments.
Daily treatments (5 days per week) were initiated with infrared light, 890 nm, 20 Joules/cm2, 20 min per treatment. After
three weeks radiographic and clinical assessment showed complete healing. 6
Study conducted by Maawan Khadra7 et all, 2004, The aim of this in vitro study was to investigate the effect of low-level
laser therapy (LLLT) on the attachment, proliferation, differentiation and production of transforming growth factor-X1
(TGF-b1) by human osteoblast-like cells (HOB). Cells derived from human mandibular bone were exposed to Ga-Al-As
diode- laser at dosages of 1.5 or 3 J/cm2 and then seeded onto titanium discs. Non-irradiated cultures served as controls.
After 1, 3 and 24 h, cells were stained and the attached cells were counted under a light microscope. In order to investigate
the effect of LLLT on cell proliferation after 48, 72 and 96 h, cells were cultured on titanium specimens for 24 h and then
exposed to laser irradiation for three consecutive days.
Specific alkaline phosphates activity and the ability of the cells to synthesize osteocalcin after 10 days were investigated
using p-nitrophenylphosphate as a substrate and the ELSA-OST-NAT immunoradiometric kit, respectively. Cellular
production of TGF-b1 was measured by an enzyme-linked immunosorbent assay (ELISA), using commercially available kits.
LLLT significantly enhanced cellular attachment.Greater cell proliferation in the irradiated groups was- observed first after
96 h. Osteocalcin synthesis and TGF-b1 production were significantly greater (Po0:05) on the samples exposed to 3 J/cm2.
However, alkaline phosphatase activity did not differ significantly among the three groups. These results showed that in
response to- LLLT, HOB cultured on titanium implant material had a tendency towards increased cellular attachment,
proliferation, differentiation and production of TGF-b1, indicating that in vitro LLLT can modulate the activity of cells and
tissues surrounding implant material.
Study conducted by Chauhan and Sarin 8 in 2006, Low level laser therapy of stress fracture of tibia in a
prospective randomized trial and found complete resolution of pain and tenderness, and return to painless
ambulation was taken as end point of therapy. Standard treatment of Stress fracture includes rest, compression,
elevation and passive stretching.
Low level laser therapy (LLLT) has been described in treatment of joint conditions, tendophaties, musculofascial pains and
dermatological conditions. 68 cases were enrolled, 34 each in control and test group. Control cases were treated with
placebo and test group with laser-therapy. Complete resolution of pain and tenderness, and return to painless ambulation
was taken as end point of therapy in both groups. The test group showed earlier resolution of symptoms and- painless
ambulation with less recurrence. LLLT appears beneficial in treatment of stress fracture in this study.
Study conducted by S.Teixeira et al,9 2006, they concluded surface characterized by a homogeneous reproducible
microtopography, microtexure and microchemistry.
Surface topographic changes induced by the laser treatment Hydroxyapatite have been of different types where compared
at different scales, as both they have produced a major enhancement on the actual surface area.
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12. The result showed that the surface topography of the substrate is attractive to the- cells, since they adhere to the cells very
strongly to the surface, being their philopodia attached between the valleys of the laser induced columnar texture.
Another study was conduct by Dimitrov et al10, 2009 , Department of Operative dentistry and Endodontics, Faculty of
Dental Medicine- Sofia, Medical University, Sofia and Department of Biochemistry, Faculty of Medicine,
Medical University, Sofia, effect of laser irradiation with different wavelength on the proliferations activity of human pul p
fibroblast cells, depending of irradiation- parameters and hard tissue thickness.
There was determined marked stimulatory effect on- proliferation activity of human pulp fibroblast cells upon direct
irradiation with infrared laser and lower upon irradiation through different sections of dental hard tissue.
Upon irradiation with Helium-neon laser was determined inhibitory effect on the proliferation activity. It‘s possible that a part
of the mesenchymal pulp cells were differentiated into another cells. That will be explained with Western blot analysis in the
second part of our investigation. Forthcoming investigations will explain the vitality of isolated mesenchymal pulp cells,
their identification and differentiation possibility, the permeability of laser beam through different sections of dentin and
enamel, power density of passed light, time and number of exposures in order to achieve the optimal effect on proliferation.
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13. Page- 13-31
2.1 Cell
2.2 Bone
2.3 Bone Fracture
2.4 Healing of fracture
2.1 Cell
The cell is the structural and functional unit of all known living organism. It is the smallest unit of an organism that is
classified as living, and is often called the building block of life. There are two types of cells: eukaryotic and prokaryotic.
Prokaryotic cells are usually independent, while eukaryotic cells are often found in multicellular organisms.
Cell Structure and functions
Each cell is a self-contained and self-maintaining entity: it can take in nutrients, convert these nutrients into
energy, carry out specialized functions, and reproduce as necessary. Each cell stores its own set of instructions
for carrying out each of these activities.
All cells share several common abilities:
 Reproduction by cell division.
 Metabolism, including taking in raw materials, building cell components, creating energy molecules and
releasing byproducts.
 Synthesis of proteins, the functional workhorses of cells, such as enzymes. A typical mammalian cell
contains up to 10,000 different proteins.
 Response to external and internal stimuli such as changes in temperature, pH or nutrient levels.
 Traffic of vesicles.
Cellular components
Figure: 2. 1.1- Eukaryotic cell components.
All cells whether prokaryotic or eukaryotic have a membrane, which envelopes the cell, separates its interior from the
surroundings, strictly controls what moves in and out and maintains the electric potential of the cell. Inside the membrane is
a salty cytoplasm (the substance which makes up most of the cell volume).
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14. All cells possess DNA, the hereditary material of genes and RNA, which contain the- information necessary to express
various proteins such as enzyme, the cell's primary machinery. Within the cell at any given time are various additional
bimolecular organelles.
The vital components of a cell are-
a) Cell membrane
b) Mitochondria –
 Cell membrane
The outer lining of a eukaryotic cell is called the plasma membrane. This membrane serves to separate and protect a cell
from its surrounding environment and is made mostly from a double layer of lipids (fat-like molecules) and proteins.
Embedded within this membrane are a variety of other molecules that act as channels and pumps, moving different
molecules into and out of the cell.
Cell membrane: Structure and Function
Structure
Figure: 2 1.2-Cell membrane
Components of the cell membrane
•It consists of two layers of phospholipids molecules.
•The head composed of protein and lipid.
•The heads are soluble in water (hydrophilic)
•The tails are insoluble in water (hydrophobic)
•They meet in the interior of the membrane.
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15. Cytoskeleton - a cell's scaffold the cytoskeleton is an important, complex, and dynamic cell component. There are a great
number of proteins associated with the cytoskeleton, each controlling a cell‘s structure by directing, bundling, and aligning
filaments. It acts to organize and maintains the cell's shape; anchors organelles in place; helps during endocytosis, the
uptake of external materials by a cell; and moves parts of the cell in processes of growth and motility.
Cytoplasm - a cell's inner space- Inside the cell there is a large fluid-filled space called the cytoplasm. This refers both to
the mixture of ions and fluids in solution within the cell, and the organelles contained in it which are separated from this
intercellular "soup" by their own membranes. The cytosol refers only to the fluid, and not to the organelles.
It normally contains a large number of organelles, and is the home of the cytoskeleton. The cytoplasm also contains many
salts and is an excellent conductor of electricity, creating the- perfect environment for the mechanics of the cell. The function
of the cytoplasm, and the organelles which reside in it, are critical for a cell's survival.
Genetic material-
Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most organisms use
DNA for their long term information storage, but a few viruses have RNA as their genetic material. The biological information
contained in an organism is encoded in its DNA or RNA sequence.
Function
1. Separates between cytoplasm & ECF.
2. Maintain cell internal environment...
3. Transport of molecules in & out the cell.
4. Controls ions distribution between cytoplasm and ECF.
5. It contains protein receptors for hormones &chemical transmitter.
6. Generates membrane potentials.
 Mitochondria– the power generators
Mitochondria are self-replicating organelles that occur in various numbers, shapes, and sizes in the cytoplasm of all
eukaryotic cells. Mitochondria play a critical role in generating energy in the eukaryotic cell. Mitochondria generate the cell's
energy by the process of oxidative phosphorylation, utilizing oxygen to release energy stored in cellular nutrients (typically
pertaining to glucose) to generate ATP. Mitochondria multiply by splitting in two.
Figure: 2.1.3- Simplified structure of mitochondrion
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16. Mitochondria-Structure and Function:
A mitochondrion contains outer and inner membranes composed of phospholipids bilayers and proteins.[6] The two
membranes, however, have different properties. Because of this double-membraned organization, there are five distinct
compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the- space
between the outer and inner membranes), the inner mitochondrial membrane, the cristae space (formed by enfolding of the-
inner membrane), and the matrix (space within the inner membrane)..The vital functional unit of mitochondrion, respiratory
chain is embedded in-between the Outer & inner membrane.
The processes that happen in the mitochondrion are
 Energy conversion
 Pyruvate oxidation,
 The Krebs cycle,
 the metabolism of amino acids, fatty acids, and steroids,
 NADH and FADH2: the electron transport chain.
 Heat production
 Storage of calcium ions
 Generation of adenosine triphosphate (ATP).
 The membrane also maintains the cell potential.
Mitochondria-Metabolism
Figure: 2 1.4-Main pathways of cellular and mitochondrial energy metabolism.
The two main metabolic pathways, i.e. glycolysis and oxidative phosphorylation are linked by the enzyme complex pyruvate
dehydrogenase. Briefly, glucose is transported inside the cell and oxidized to pyruvate. Under aerobic conditions, the
complete oxidation- of pyruvate occurs- through the TCA cycle to produce NADH, H+ and/or FADH2. In this picture (figured
in pink) the glutamine oxidation pathway is shown.
Mitochondrial respiratory chain.
Figure: 2.1.5: Schematic diagram of mitochondrial respiratory chain .
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17. The mitochondrial respiratory chain consists of four enzyme complexes (complexes I- IV) and two intermediary substrates
(coenzyme Q and cytochrome c). The NADH+H+ and FADH2 produced by the intermediate metabolism are oxidized further
by the mitochondrial respiratory chain to establish an electrochemical gradient of protons, which is finally used by the F1F0-
ATP synthase (complex V) to produce ATP, the only form of energy used by the cell.
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18. 2.2- Bone
General aspects of bone
At the molecular level, bone is one of the few materials in the body that contains a mineral-like component in addition to the
organic components, others include dentin and enamel.
Bone is a composite material, which consists of organic matrix (mainly collagen) and inorganic hydroxyapatite (HA). Water
accounts for about 20% of the wet weight of cortical bone, HA makes up approximately 45%, and organic substances
account for the remaining 35%. Cortical bone, which surrounds all bones, primarily serves the supportive and- protective
function of bone, whereas trabecular (cancellous) bone is mostly responsible for the metabolic function.
Functions of Bones
o Mechanical Protection — Bones can serve to protect internal organs, such as the skull protecting the brain or the
ribs protecting the heart and lungs.
o Shape — Bones provide a frame to keep the body supported.
o Movement — Bones, skeletal muscles, tendons, ligaments and joints function together to generate and transfer
forces so that individual body parts or the whole- body can be manipulated in three-dimensional space.
o Sound transduction — Bones are important in the mechanical aspect of overshadowed hearing.
o Synthetic
o Blood production — The marrow, located within the medullary cavity of long bones and interstices of cancellous
bone, produces blood cells in a process called haematopoiesis.
o Metabolic
o Mineral storage — Bones act as reserves of minerals important for the body, most notably calcium and
phosphorus.
o Growth factor storage — Mineralized bone matrix stores important growth factors such as insulin-like growth
factors, transforming growth factor, bone morphogenetic proteins and others.
o Fat Storage — the yellow bone marrow acts as a storage reserve of fatty acids
o Acid-base balance — Bone buffers the blood against excessive pH changes by absorbing or releasing alkaline
salts.
o Detoxification — Bone tissues can also store heavy metals and other foreign elements, removing them from the
blood and reducing their effects on other tissues. These can later be gradually released for excretion. [Citation
needed]
o Endocrine organ - Bone controls phosphate metabolism by releasing fibroblast growth factor - 23 (FGF-23), which
acts on kidney to reduce phosphate reabsorption.
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19. Types of Bone (General)
Table: 2.2.6-Schematic diagram of bone structure
 Types of Bone(Macroscopic)
Figure: 2.2 .7-A femur head with a cortex of compact bone and medulla of trabecular bone.
Bone in human bodies is generally classified /categorized morphologically into two types, 1) Cortical bone, also known as
compact bone and 2) Trabecular bone, also known as cancellous- or spongy bone. These two types (Compact &
Trabecular) are classified as on the basis of porosity and the unit microstructure. .Trabecular bone accounts for 20% of total
bone mass but has nearly ten times the surface area of compact bone. Compact bone is, as the name suggests, a
compacted and stiff material with- a relatively low porosity. Trabecular bone is a more porous structure comprised of small
struts and plates called-
Trabecular. Cortical bone is much denser with a porosity ranging between 5% and 10%% while the porosity of trabecular
bone ranges from 50% to 90%. Cortical bone is found primary is found in the shaft of long bones and- forms the outer shell
around cancellous bone at the end of joints and the vertebrae. Trabecular bone is found in the medullary cavity of flat and
short bones, and in the epiphysis and metaphysis of long bones. At the tissue level, it is thought that the two bone types are
identical.
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20. Types of Bone (Microscopic)
Lamellar Bone Woven Bone
Figure 2.2.8a - Lamellar Bone & 2.28b- Woven Bone
 Lamellar Bone: Lamellar bone is stronger and filled with many collagen fibers parallel to other fibers in the same
layer (these parallel columns are called osteons).
 Woven Bone (non-lamellar): Woven bone is weaker, with a small number of randomly oriented fibers, but forms
quickly. It is replaced by lamellar bone, which is highly organized in concentric sheets with a low proportion of
osteocytes.
Bone Composition
Table: 2.2.9 – Schematic diagram of bone composition
 Cells
Osteoprogenitor cells
o
Osteoclasts
o
Osteocytes
o
Osteoclasts
o
 Extracellular Matrix
Organic (35%)
o Collagen (type I) 90%
o Osteocalcin, osteonectin, proteoglycans, glycosaminoglycans, lipids (ground substance)
Inorganic (65%)
o Primarily hydroxyapatite Ca5(PO4).
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21. Cells of Bone:
There are 4 types of cells constituting the bone-
 Osteoprogenitor cells
The osteoprogenitor cell is a primitive cell derived from the mesenchyme. It forms in the inner layer of the periosteum
and lines the marrow cavity as well as Haversian and Volkmann‘s canals of compact bone. During periods of growth
and remodeling these cells are stimulated to differentiate into osteoblasts that lay down new bone. They can also
differentiate into other cell types such as fibroblasts, chondroblasts and adipose cells during bone loss . In mature bone
that is not actively remodeling, these cells are quiescent and are called bone- lining cells. Bone lining cells are
generally inactive and have very few cytoplasmic organelles. Their processes extend through canaliculi to neighboring
cells which suggests that they may be involved in mechano-transduction and cellular communication.
 Osteoblasts
Osteoblasts are cells which are responsible for the production of organic bone matrix. These cells synthesize and
secrete small vesicles into the existing bone. These matrix vesicles are formed by pinching off portions of the
plasmalemma and contain enzymes, including alkaline phosphatase, which load the vesicle with calcium 8. Rupture of
these vesicles initiates local mineralization by releasing calcium and by- negating local inhibiting mechanisms.
Deposition of mineral makes the bone matrix stiffer, impermeable and more capable of bearing loads.
 Osteocytes
Osteocytes are the most abundant cells in the bone matrix and are mature osteoblasts that have been ‗walled-in‘ by the
bone tissue which has been laid down around them.
Approximately 10% of all active osteoblasts are converted into osteocytes. The full role of these cells is still not known
however, they are thought to have mechanosensory and chemosensory regulatory roles. Osteocytes are a candidate
mechanosensory cell type because- they are ideally situated to sense mechanical stimulation such as strain or
interstitial fluid- flow.These actions are caused by mechanical loading and thus osteocytes are thought to be in some
way responsible for bone adaptation and remodeling9. Osteocytes maintain healthy tissue by secreting enzymes and
controlling the bone mineral content, they also control the calcium release from the bone tissue to the blood.
 Osteoclasts
Osteoclasts are large multinucleate cells that break down bone tissue. They are derived from the mononuclear
phagocytic lineage of the haemopoietic system. They are formed by monocytes either by the fusion of several cells or
by DNA replication without cell division in response to stimuli from osteoblasts, osteocytes and hormones. When these
cells are active- they rest directly on the bone surface in a resorption bay or a Howship‘s lacuna. They are
characterized by two easily identifiable features; the ‗ruffled border‘ which is an infolded plasma membrane where the
resorption takes place, and the ‗clear zone‘ which is the point of attachment of the osteoclast to the underlying bone
matrix 10.
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22. Extracellular Matrix
Matrix comprises a major constituent of bone. It consists of living cells embedded in a calcium carbonate matrix that
makes up the main bone material. The majority of bone is made of the bone matrix & bone cell. In mature bone, 10-
20% by weight of the matrix is water. Dried bone consists of about 70% inorganic matrix and 30% organic matrix by
weight. The organic matrix is 90-95% collagen fibers with the remainder being a homogenous ground substance.
Figure: 2.210-Picture showing Bone composition & extracellular-
matrix.
1. Organic
The organic part of matrix is mainly composed of Type I collagen. This is synthesized intracellularly as tropocollagen
and then exported, forming fibrils. The organic part is also composed of various growth factors, the functions of which
are not fully known.
Factors present include glycosaminoglycans, osteocalcin, osteonectin, bone sialo protein, osteopontin and Cell
Attachment Factor. One of the main things that distinguishes the matrix of a bone from that of another cell is that the
matrix in bone is hard. In the event of a broken bone, the cells are brought out of semi-stasis to repair the matrix.
2. Inorganic
The inorganic part is mainly composed of crystalline mineral salts and calcium, which is present in the form of
hydroxyapatite. The matrix is initially laid down as unmineralised osteoid (manufactured by osteoblasts). Mineralization
involves osteoblasts secreting vesicles containing alkaline phosphatase. This cleaves the phosphate groups and acts
as the foci for calcium and phosphate deposition. The vesicles then rupture and act as a centre for crystals to grow on.
Osteoblasts, cells that take up calcium compounds from the blood and secrete sturdy bone matrix, live on the surface
of existing matrix. Cells gradually become embedded in their own matrix, forming uncalcified bone matrix (osteoid). The
addition of calcium phosphate forms the calcified bone matrix, which surrounds the mature bone cells (osteocytes).
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23. Anatomy of bone
Figure: 2.2.11- Parts of a long bone.
Long bones are characterized by a shaft, the diaphysis that is much greater in length than width. They are comprised mostly
of compact bone and lesser amounts of marrow, which is located within the medullary cavity, and spongy bone.
Most bones of the limbs, including those of the fingers and toes, are long bones. The exceptions are those of the wrist, ankle
and kneecap. Short bones are roughly cube-shaped, and have only a thin layer of compact bone surrounding a spongy
interior.
The bones of the wrist and ankle are short bones, as are the sesamoid bones. Flat bones are thin and generally curved, with
two parallel layers of compact bones sandwiching a layer of spongy bone. Most of the bones of the skull are flat bones, as is
the sternum. Irregular bones do not fit- into the above categories. They consist of thin layers of compact bone surrounding a
spongy interior. As implied by the name, their shapes are irregular and complicated. The bones of the spine and hips are
irregular bones. Sesamoid bones are bones embedded in tendons. Since they act to hold the tendon further away from the
joint, the angle of the tendon is increased and thus the leverage of the muscle is increased. Examples of sesamoid bones
are the patella and the pisiform. The point where two or more bones come together is called a joint, or articulation. Different
kinds of joints enable different ranges of motion. Some joints barely move, such as those between the interlocking bones of
the skull. Other bones, held together by tough connective- tissues called ligaments, form joints such as the hinge joint in the
elbow, which- permits movement in only one direction. The pivot joint between the first and second vertebrae allows the
head to turn from side to side.
Intimately associated with bone is another type of connective tissue called cartilage. Cartilage is softer, more elastic, and
more compressible than bone. It is found in body parts that require both stiffness and flexibility, such as the ends of bones,
the tip of the nose, and the outer part of the ear. Bone is not a uniformly solid material, but rather has some spaces between
its hard elements.
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24. Figure: 2.2.12- a, b, c, d- Appendicular upper extremity bones.
Figure: 2.2.12- e, f, g- Appendicular lower extremity bones.
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25. Histology of bone
Figure: 2.2.13 a-Schematic histological structure of bone.
Haversian system (osteon), functional unit of bone-
Figure: 2.2.13b: HaversianCanal: A higher magnification view of slide clearly shows the concentric circles.
After osteoclasts remove old bone, osteoblasts deposit bone in this circular arrangement beginning with
the outer ring and working inward. As the osteoblasts become trapped in their own calcified deposits, they
are known as osteocytes.
Haversian canal carries blood vessel through center of osteon lamellae "little layer" of matrix between concentric rings of
osteocytes lacunae "pools" which house osteocytes osteocytes "bone cells" which- maintain bone- Volkmann's canal feeder
cross connecting vessel for blood supply- canaliculi protoplasmic extensions from osteocytes by which maintenance of bone
is performed, interstitial lamellae layers between adjacent Haversian systems. Osteoblasts form the lamellae sequentially,
from the most external inward toward the Haversian canal. Some of the osteoblasts develop into osteocytes, each living
within its own small space, or lacuna. Osteocytes make contact with the cytoplasmic processes of their counterparts via a
network of small canals, or canaliculi. This network facilitates the exchange of nutrients and metabolic waste.
Collagen fibers in a particular lamella run parallel to each other but the orientation of collagen fibers within other lamellae is
oblique. The collagen fiber density is lowest at the- seams between lamellae, accounting for the distinctive microscopic
appearance of a transverse section of osteons.
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26. 2.3 Bone Fracture
A bone fracture is a break in a bone. Most people fracture at least one bone during their lifetime. The
severity of fractures increase with age. Children's bones are more flexible and less likely to break. Falls or
other accidents that do not harm children can cause complete fractures in older- adults. Older adults suffer
from fractures more than children because their bones are more likely to be brittle.
Causes of fracture
Fractures occur when a bone can't withstand the physical force exerted on it. Fractures happen because an area of bone is
not able to support the energy placed on it (quite obvious, but it -becomes more complicated). Therefore, there are two
critical factors in determining why a fracture occurs:
 The energy of the event
 The strength of the bone
The energy can being acute, high-energy (e.g. car crash), or chronic, low-energy (e.g. stress fracture). The bone strength
can either be normal or decreased (e.g. osteoporosis). A very simple problem, the broken bone, just became a whole lot
more complicated!
Mechanism of Bone fracture
a. Tension,
b. Compression,
c. Bending, shear,
d. Torsion &
e. Combined effects
Types of Fractures
Figure: 2.3.14-Mechanism of bone fracture Figure: 2.3.15-Different types of fracture.
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27. Symptoms of a fracture
The most common symptoms of a fracture are:
 Swelling around the injured area
 Loss of function in the injured area
 Bruising around the injured area
 Deformity of a limb.
2.4- Healing of fracture
Figure: 2.4.16-Schematic diagram of fracture healing
The natural process of healing a fracture starts when the injured bone and surrounding tissues bleed, forming what's called
fracture Hematoma. The blood coagulates to form a blood clot situated between the broken fragments. Within a few days
blood vessels grow into the jelly-like matrix of the blood clot. The new blood vessels bring phagocytes to the area, which
gradually remove the non-viable material.
The blood vessels also bring fibroblasts in the walls of the vessels and these multiply and produce collagen fibers. In this
way the blood clot is replaced by a matrix of collagen. Collagen's rubbery consistency allows bone fragments to move only a
small amount unless severe or persistent force is applied.
At this stage, some of the fibroblasts begin to lay down bone matrix (calcium hydroxyapatite) in the form of insoluble
crystals. This mineralization of the collagen matrix stiffens it and transforms -it into bone. In fact, bone is a mineralized
collagen matrix; if the mineral is dissolved out of bone, it becomes rubbery.
This initial "woven" bone does not have the strong mechanical- properties of mature bone. By a process of remodeling, the
woven bone is replaced by mature "lamellar" bone. The whole process can take up to 18 months, but in adults the strength
of the healing bone is usually 80% of normal by 3 months after the injury. Healing bone callus is on average sufficiently
mineralized to show up on X-ray within 6 weeks in adults and less in children.
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28. Phases of fracture healing
Table: 2.4.17- schematic diagram of Phases of fracture healing.
Bone Healing
There are three major phases of fracture healing, two of which can be further sub-divided to make a total of five phases;
1. Reactive Phase
i. Impact, Induction and inflammation.
ii. Granulation tissue formation
2. Reparative Phase
iii.Callus formation
iv.Lamellar bone deposition
3. Remodeling Phase
v. Remodeling to original bone contour.
Reactive
After fracture, the first change seen by light and electron microscopy is the presence of blood cells within the tissues which
are adjacent to the injury site. Soon after fracture, the blood vessels constrict, stopping any further bleeding.[1] within a few
hours after fracture, the extra-vascular- blood cells, known as a "hematoma", form a blood clot. All of the cells within the
blood -clot degenerate and die.[2] Some of the cells outside of the blood clot, but adjacent to the injury site, also degenerate
and die.[3] Within this same area, the fibroblasts survive and replicate. They form a loose aggregate of cells, interspersed
with small blood vessels, known as granulation tissue.[4].
Reparative
Days after fracture, the cells of the periosteum replicate and transform. The periosteal cells proximal to the fracture gap
develop into chondroblasts and form hyaline cartilage. The periosteal cells distal to the fracture gap develop into osteoblasts
and form woven bone.
The fibroblasts within the granulation tissue also develop into chondroblasts and form hyaline cartilage. These two new
tissues grow in size until they unite with their counterparts from- other pieces of the fracture. This process forms the fracture
callus.[6] Eventually, the fracture gap is bridged by the hyaline cartilage and woven bone, restoring some of its original
strength.
The next phase is the replacement of the hyaline cartilage and woven bone with lamellar bone. The replacement process is
known as endochondral ossification with respect to the hyaline cartilage and "bony substitution" with respect to the woven
bone. Substitution of the woven bone- with lamellar bone precedes the substitution of the hyaline cartilage with lamellar
bone.
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29. The lamellar bone begins forming soon after the collagen matrix of either tissue becomes mineralized. At this point,
"vascular channels" with many accompanying osteoblasts penetrate the mineralized matrix.
The osteoblasts form new lamellar bone upon the recently exposed surface of the mineralized matrix. This new lamellar
bone is in the form of trabecular bone.[7] Eventually, all of the woven bone and cartilage of the original fracture callus is
replaced by trabecular bone, restoring most of the bone's original strength.
Remodeling
The remodeling of bone requires the coordinated activity of two types of cells:
 Osteoclasts that demineralise bone in their vicinity
 Osteoblast that secretes collagen and mineral to lay down new bone.
Stages / Cycle of Bone Remodeling:
1. Resting,
2. Resorption,
3. Reversal,
4. Bone formation
Figure: 2.4.18-Schematic diagram of Bone Remodeling
Normal bone remodeling. (i) Resorption: stimulated osteoblast precursors release factors that induce osteoclast
differentiation and activity. Osteoclasts remove bone mineral and matrix, creating an erosion cavity. (ii) Reversal:
mononuclear cells prepare bone surface for new- osteoblasts to begin forming bone. (iii) Formation: successive waves of
osteoblasts synthesize an organic matrix to replace resorbed bone and fill the cavity with new bone. (iv) Resting: bone
surface is covered with flattened lining cells. A prolonged resting period follows with little cellular activity until a new
remodeling cycle begins. Bone remodeling is a lifelong process where old bone is removed from the skeleton (a sub-process
called bone resorption) and new bone is added (a sub-process called ossification or bone formation). In the first year of life,
almost 100% of the skeleton is replaced. In adults, remodeling proceeds at about 10% per year.[1] Histological analysis of
secondary fracture healing in bone showing the progression of repair on days 1, 3, 14, 21, and 28.
Fractured bone appears denser than the surrounding tissue. On day 7, extensive soft callus is seen forming around the
injured bone. At day 14, the soft callus becomes mineralized to form new bone and achieve union by day 21 and 28 (H&E
stain, x40)20.
In the process of fracture healing, several phases of recovery facilitate the proliferation and protection of the areas
surrounding fractures and dislocations. The length of the process depends on the extent of the injury, and usual margins of
two to three weeks are given for the reparation of most upper bodily fractures; anywhere above four weeks given for lower
bodily injury.
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30. The process of the entire regeneration of the bone can depend on the angle of dislocation or fracture. While the bone
formation usually spans the entire duration of the healing process, in some instances, bone marrow within the fracture
having- healed two or fewer weeks before the final remodeling phase. While immobilization and surgery may facilitate-
healing, a fracture ultimately heals through physiological processes. The healing process is mainly determined by the-
periosteum (the connective tissue membrane covering the bone).
The periosteum is the primary source of precursor cells which develop into- chondroblasts and osteoblasts that are essential
to the healing of bone. The bone marrow (when present), endosteum, small blood vessels, and fibroblasts are secondary
sources of precursor cells.
Factors which affects fracture healing
Fracture treatment is not purely a question of effective fracture reduction and fixation but a complex biological process. The
natural tendency for a fracture is to unite. When delay or failure of union occurs, the causes are either local factors at the
site of fracture or defects in the methods employed in treatment.
Figure: 2.4.19- Schematic diagram of bone healing factors.
 General Causes
a) Imperfect immobilization:
(I) Too little extent of immobilization. And
(ii) Too short a period of immobilization.
b) Distraction: Too heavy a pull of the distal fragment by skeletal traction.
c) Surgical intervention: This empties the fracture hematoma and strips the periosteum, interfering with the blood supply and slowing the
healing process.
 Local causes
a) Infection: This is the commonest cause for delayed union or non-union in open fractures.
b) Inadequate blood supply to one fragment: Certain sites are notorious for slow union or non-union e.g. (I) Fracture neck of femur. The
blood supply to the head of the femur is poor.
(ii) Fracture scaphoid. The blood supply to the proximal fragment is poor.
c) Interposition of soft tissues between the fragments prevents bony apposition and interferes with healing.
d) Type of fracture: Transverse fractures unite slowly compared to oblique or spiral fractures.
e) Type of bone: Fracture at the cancerous ends of bone unites better than those in the mid shaft of long bones where cancellous bone is
minimal.
Fractures in children unite very rapidly whereas delayed union is common in the aged. Other factors like protein and vitamin deficiencies,
general diseases like syphilis and diabetes play only a small part in influencing the rate of healing. Bio-Compression at the fracture site
through protected weight bearing at the proper time promotes healing of the fractures.
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31. Although there are no magical ways to fix a bone fracture, but there are ways to help speed up the
healing process, and help fracture to heal properly/ faster.
 Proper medical management.
 Nutrition Support.
 Osteoblast cell injections
 Electrical stimulation.
 Magnetic stimulation.
 Ultrasound therapy.
 Gene-therapy
 Low Level Laser therapy.
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32. Page- 32-42
3.1 Laser
3.2 Laser principle
3.3 Components of a laser system
3.4 Laser Machine
3.5 Measurement, Parameter & Protocol of Laser
3.1 Laser
“Any device which can be made to produce or amplify electromagnetic radiation in the
wavelength range from 180nm to 1mm primarily by process of controlled stimulated
emission” European Standard 1EC 601 .
Criteria of Low Level Laser
 Coherent-referring to the wave nature of light, the peaks and troughs of the waves occur synchronously in time (i.e., a
fixed phase relationship between the electric fields of the electromagnetic field)
 Collimated-exhibiting minimal divergence (increase in the beam diameter) as the beam propagates
 Monochromatic-of a single or very limited spectral line width, i.e., a single color
 High intensity-displaying a high optical power per unit area for a given amount of energy compared to broadband
sources
Types and Classification of Lasers: Lasers have been classified with respect to their hazards based on power,
wavelength, and pulse duration. These definitions are wordy and cumbersome to read out of context, but when given the
specifications of a laser or laser systems are not difficult to apply.
 Types
 According to their sources:
Gas Lasers
Crystal Lasers
Semiconductors Lasers
Liquid Lasers
 2. According to the nature of emission:
Continuous Wave
Pulsed Laser
Q-switched lasers
while most often laser types are discussed in terms of what they can treat, it is important to
recognize the broader categories of lasers.
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33.  Continuous wave (cw) lasers
A continuous beam and include these emit the CO2 and krypton lasers. Pseudo-continuous wave lasers emit a beam in
such close pulses that the effect on tissue is similar to that of a continuous wave laser. These lasers are used to coagulate
tissue, as for example in the treatment of moles and warts.
 Pulsed lasers
Lasers that emit a beam in short pulses usually separated by 0.1-1 second. Pulsed lasers are more selective in their
destructive effect than continuous wave lasers, and are used in selective photothermolysis.
 Q-switched lasers
Q-switching refers to the process of storing up laser energy in the laser cavity and releasing it in one single very short and
extremely powerful pulse. This results in power outputs in the megawatt to gig watt range, and allows for mechanical (vs.
thermal) destruction of the target. Such lasers are often used in the removal of tattoos.
 3. According to their wavelength:
Visible Region
Infrared Region
Ultraviolet Region
Microwave Region
X-Ray Region
Table:3.1.1- Types of laser according to wavelengths
Classes of Lasers (adoptedfromANSIZ-136.1-2000) -Class Levels 1-4:
• 1 = incapable of producing damaging radiation levels (laser printers & CD players)
• 2 = low-power visible lasers (400-700 nm wavelength, 1 mW)
• 3 = medium-power lasers - needs eye protection
• 3a – up to 5 mW
• 3b** – 5 mw-500 mW
• 4 = high-power lasers– presents fire hazard (exceeds 500 mW).
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34. 3.2 Laser principle-
Stimulated emission is fundamental to light amplification and thus to the operation of the laser. To understand it, it must
be placed in the context of interactions between light and matter. Here, the matter is composed of optically active
elements in ―solution‖ in a gas, plasma, solid or liquid medium. These elements can be atoms, ions, molecules, free
radicals or electrons (for simplicity, we consider ―atoms‖ in the following). Their energy levels are quantified and are such
that light of a certain frequency can interact with the population found in these levels. More precisely, let us consider two
energy levels E1 and E2 (E1 is less than E2) whose atoms can interact with light of frequency . The
group E1-E2 is called radiative transition if atoms can only pass from E 1 to E2 (or from E2 to E1) by interacting with light.
E1 is called the lower energy level and E2 the upper energy level.
The emission-absorption principle
The three different mechanisms are shown below (Figure 3.2.2):
1. Absorption: An atom in a lower level absorbs a photon of frequency hν and moves to an upper level.
2. Spontaneous emission: An atom in an upper level can decay spontaneously to the lower level and emit a photon of
frequency hν if the transition between E2 and E1 is radiative. This photon has a random direction and phase.
3. Stimulated emission: An incident photon causes an upper level atom to decay, emitting a ―stimulated‖ photon whose
properties are identical to those of the incident photon. The term ―stimulated‖ underlines the fact that this kind of radiation
only occurs if an- incident photon is present. The amplification arises due to the similarities between the incident and
emitted photons.
Figure 3.2.2: Mechanism of the interaction between an atom and a photon (The photon has
an energy hν equal to the difference between the two atomic energy levels).
Competition between the three mechanisms
For a radiative transition, these three mechanisms are always present at the same time. To make a laser medium,
conditions have to be found that favour stimulated emission over absorption and spontaneous emission. Thus, both the
right medium and the right conditions must be chosen to produce the laser effect. An incident photon of energy has
an equal chance of being absorbed by a ground-state atom as being duplicated (or amplified!) by interacting with an
excited-state atom. Absorption and stimulated emission are really two reciprocal processes subject to the same
probability. To favour stimulated emission over absorption, there need to be more excited-state atoms than ground-state
atoms. Spontaneous emission naturally tends to empty the upper level so this level has to be emptied faster by-
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35. stimulated- emission. It has been proved that stimulated emission is much more likely to happen if the medium used is-
flooded with light (i.e. with a large number of photons). A good way to do this is to confine the photons in an optical
cavity.
Population inversion and pumping
If there are more atoms in the upper level (N 2) than in the lower level (N1), the system is not at equilibrium. In fact, at
thermodynamic equilibrium, the distribution of the atoms between the levels can be given by Boltzmann's Law.
N2= N1x exp – {(E2-E1)/ KT}.
In this case, N2 is always less than N1. A situation not at equilibrium must be created by adding energy via a process
known as ―pumping‖ in order to raise enough atoms to the upper level. This is known as population inversion and is
given by . Light is amplified when the population inversion is positive. Pumping may be electrical,
optical or chemical.
Spectroscopic systems used to create a laser
Not all atoms, ions and molecules, with their different energy levels, are capable of creating a population inversion and a
laser effect. Only radiative transitions (where the atoms are excited due to light absorption) should be used and non-
radiative transitions should be avoided. Some transitions have both a radiative and a non-radiative part. In this case, the
upper level empties as a result of a non-radiative effect as well as spontaneous emission. This leads to additional
problems for achieving a population inversion because it is difficult to store atoms in the upper level under these
conditions. Thus, this type of transition should also be avoided.
Next, the relative energy levels specific to each type of atom must be considered. For example, choosing a lower level
with more energy than the ground state will greatly limit the population N 1, which may even be zero (Figure 3). This
means that only one atom would have to be excited to achieve population inversion.
Figure 3.2.3: Laser transition with the lower level far above the ground state.
The population at thermodynamic equilibrium is defined by Boltzmann's Law.
In addition, pumping must be able to move atoms to a higher level. Every pumping system (particularly optical or
electrical) corresponds to a certain energy, which must be transferable to the atoms of the medium. The difference in
energy between the excited state and the ground state must match the pumping energy. In optical pumping, there must
be at least three different- energy levels to create a population inversion. Figure 4 illustrates such a system. It shows the-
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36. pumping transition- (between E1 and E3) and the laser transition (between E2 and E1). The objective is to store atoms in
level E2 by absorbing ―pumping‖ radiation whose wavelength is shorter than that of the laser transition. This means that
the excited atoms must quickly decay from level 3 to level 2 only, a condition that limits the choice of systems that will
work. Figure 4 also shows an ideal cycle for an atom: it rises into level 3 by absorbing a photon from- the pumping light. It
then falls very- rapidly into level 2. Finally, it decays by stimulated emission to level 1.
Despite its simplicity, this is not a very easy system to implement as the ground state of the laser transition has a large
population at thermodynamic equilibrium and at least half of this population must be excited to level 2 to obtain
population- inversion. Moreover, level 2 must be able to store these atoms so spontaneous emission must be- very
unlikely. This affects the choice of the system. A large pumping energy is also needed. The first ever laser was of this -
type and used a ruby (Cr3+:Al2O3). Ruby is composed of an aluminium crystal matrix and a doped ion (Cr 3+) whose
energy levels are used to create the laser effect. The medium is strongly pumped by discharge lamps.
Figure 3.2.4: Example of a three-level system with optical pumping.
Another example of a spectroscopic system is the four-level laser (Figure 5). Here, the pumping transition (optical
pumping) and the laser transition occur over a pair of distinct levels (E 0 to E3 for the pump and E1 to E2 for the laser).
E1 is chosen to be sufficiently far from the ground state E 0 so that the thermal population at thermodynamic equilibrium
is negligible. Similarly, atoms do not stay in level 3 or level 1. Figure 5 represents an ideal four-level system. Unlike- the
three-level system, as soon as one atom moves to level 2, a population inversion occurs and the medium becomes
amplifying. To maintain the population inversion, atoms must not accumulate in level 1 but must rapidly decay to level 0.
One of the best known mediums operating in this way is neodymium YAG (Nd 3+:Y3Al5O12).
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37. Figure3.2 5: Example of a four-level system with optical pumping.
A final example of a spectroscopic system providing a laser effect is the helium-neon gas system (Figure 6). In this case
the pumping method is electrical. Neon transitions are used for the laser transitions: there are several but the most well-
known is the coloured one at 632.8 nm. Helium is used as an intermediary gas, capable of transferring energy from the
electrons to the neon particles via collisions. Helium is also unique in having two excited states said to- be ―metastable‖
i.e-. atoms can stay- there a long time before falling to the ground state. Helium atoms are carried into- the excited state
by collisions with electrons.
Energy is easily transferred to neon when the atoms collide because these metastable levels coincide with the excited
states of neon. This process is given by the equation: He* + Ne -> He +Ne*
An excited helium atom meets a ground-state neon atom and transfers its energy while decaying.Figure 3.2.6 also shows
that the lower levels of the laser transitions are far from the ground state, which favours population inversion (no thermal
population).
Figure 3.2.6: A Helium-Neon Laser System.
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38. 3.3. Components of a laser system
Laser-Components
Figure: 3.3.7: Diagram of Components of laser System
All lasers are composed of four basic components:
o The lasing medium
o The optical cavity
o The pumping system
o The delivery system
There are three different vital parts to a Ga-Al-As laser: 1. an energy source, 2. a laser material that absorbs this energy
emits it as light, and 3. a cavity that makes the light resonate and channels it in to narrow beam. Within the cavity very high
circulating photon densities stimulate the emission of light from the energized laser material. This design creates a powerful
beam of billions of photons, unlike to laser and differentiating them from lower intensity light sources like LEDs.
3.4 Laser Machine
Figure: 3.4.8: schematic diagram of interior of a laser machine.
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39. There are five (5) basic components that make up the laser system, the control panel, the motherboard, the DC power
supply, the laser tube assembly, and the motion system.
A. DC Power Supply.
B. Motherboard
C. Control Panel
D. Laser Tube Assembly
E. Motion System
Figure: 3.4.9: schematic diagram of the control panel of a laser machine
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40. 3.5 Measurement, Parameter & Protocol of Laser
3.5.1 Calculating Laser and Treatment Parameters-
Laser Therapy devices are generally specified in terms of the average output power (milliwatts) of the laser diode,
and the wavelength (nanometers) of light they emit. This is necessary information, but not enough with which to
accurately define the parameters of the laser system. To do this, one must also know the area of the laser beam
(cm 2) at the treatment surface (usually the tip of the hand piece when in contact with the skin).
If the output power (mW) and beam area (cm 2) are known, it is a reasonably straight-forward exercise to calculate
the remaining parameters which allow the precise dosage measurement and delivery.The output power of a laser,
measured in milliwatts, refers to the number of photons emitted at the particular wavelength of the laser diode.
Power Density measures the potential thermal effect of those photons at the treatment area. It is a function of Laser
Output Power and Beam area, and is calculated as:
Laser Output Power (W)
1) Power Density (W/cm2) =
Beam area (cm2)
Beam area can be calculated by either:
2) Beam Area (cm 2) = Diameter(cm)2 x 0.7854
or: Beam Area (cm2) = Pi x Radius(cm)2
The total photonic energy delivered into the tissue by a laser diode operating at a particular output power ov er a certain period is measured in
Joules, and is calculated as follows:
Laser Output Power (Watts) x Time (Secs)
3) Energy (Joules) =
It is important to know the distribution of the total energy over the treatment area, in order to accurately measure
dosage. This distribution is measured as Energy Density (Joules/cm 2). "For a given wavelength of light, energy
density is the most important factor in determining- the tissue reaction"(Baxter, 1994). Energy Density is a function
of Power Density and Time in seconds, and is calculated as:
Laser Output Power (Watts) x Time (Secs)
4) Energy Density (Joule/cm2) =
Beam Area (cm2)
OR: Energy Density (Joule/cm2) = Power Density (W/cm 2) x Time (Secs)
To calculate the treatment time for a particular dosage, you will need to know the Energy Density (J/cm 2 ) or Energy (J), as well as the Output
Power (mW), and Beam Area (cm 2 ). First, calculate the Output Power Density (mW/cm 2 ) as per Equation 1, then:
Energy Density (Joules/cm2)
5) Treatment Time (Seconds) = Output Power Density (W/cm2)
Energy (Joules)
or: Treatment Time (Seconds) =
Laser Output Power (Watts)
Finally:
Laser Output Power (mW)
Laser Output Power (Watts) =
1000
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41. Output Beam Treatment Energy Energy Density
Power Spot Size Time (Secs) (Joules) (Joules/cm 2)
(mW) (cm 2)
5 0.1 8.0 0.04 0.4
50 0.1 8.0 0.4 4.0
125 0.2 8.0 1.0 5.0
250 0.2 8.0 2.0 10.0
500 0.2 8.0 4.0 20.0
Table: 3. 5.10: Various Laser Parameters v Dosage/Time: Illustrates the
difference in Joules and Joules/cm 2 dosages for differing output parameters.
The calculation of these parameters is explained above.
3.5.2 Laser Parameters for Effective Treatment
"For a given wavelength of light, energy density is the most important factor in determining the tissue reaction"
(Baxter, 1994). Research indicates that Energy Densities in the range 0.5 to 4 Joules/cm 2 are most effective in
triggering a photobiological response in tissue (e.g. Mester & Jaszagi-Nagy, 1973; Mester & Mester, 1989; Mashiko
et al, 1983; Haina, 1982), with 4 Joules/cm 2 having the greatest effect on wound healing (Mester et al, 1973; Mester
et al, 1989).
Australian research suggests that this 'therapeutic window' of biostimulation may be extended to include
10/Joules/cm 2 (Laakso et al, 1994), and has applications in other areas of practice, such as Myofascial Trigger Point
therapy and pain control and tissue healing. Dosages above 10J/cm 2 is proved to be bioinhibitive, and the resulting
bioinhibition, may also have therapeutic applications, such as in the treatment of keloid scarring and pain
management. Many practitioners have found straight Joules dosages - up to 20 J/cm 2 in some cases 94.7J/cm2, to
be effective in the treatment of a number of common musculoskeletal disorders. This is possibly due to the
combined action of the pain attenuating properties of laser bioinhibition at high dosages, and the biostimulatory
effect of the lower-powered 'halo' around the target treatment point. However, the same effect may not be elicited
from a different laser unit, due to differences in laser parameters (esp. Power Density) and configuration.
It is the Output Power Density which determines the time required delivering a particular Energy Density
(Joules/cm 2) dosage, and the Output Power which determines the corresponding Energy (Joules) delivered during
that time. Results obtained from particular dosages and treatments are likely to vary between individual pr actitioners
and patients, therefore, practitioner discretion is recommended in determining the applicable wavelength and -
dosage parameters for- each patient. It is important to note- that the appropriate- configuration of a laser unit will
depend primarily upon the types of conditions most commonly treated, and so specific requirements will generally
differ between practitioners.
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42. 3.5.3 Treatment Protocol, Frequency, and Response
To maximize irradiance at the target tissue, the laser probe should be held in contact with, and perpendicular to, the
tissue surface. When treating open wounds, the probe should be held slightly away from the tissue surface, whilst
still maintaining a 90 o angle. The probe tip may be covered with plastic cling film, in order to redu ce the likelihood of
cross-contamination.
In treating musculoskeletal conditions, laser therapy should be carried out following cryotherapy as the
vasoconstriction caused by cooling the tissue will increase the penetration depth of the laser irradiation. Laser
therapy helps to relax muscles, and so manipulations should be carried out following laser irradiation. Heat
therapies and various creams and lotions can be applied after laser therapy.
Laser treatments can be carried out by irradiating daily for the first week, then gradually increasing the interval
between treatments over successive weeks, according to the progression of the condition being treated. The total
dosage should not exceed 100-200 J in any single treatment session.
Laser dosage is cumulative, and so overtreatment causing a degradation of LLLT effectiveness can come from
overly-high dosages in one treatment session, or too many treatment sessions in close succession. Individual
practitioner discretion is to be used to determine the appropri ate maximum session dosage, and the frequency of
treatment, for each particular patient.
Patients may report a number of sensations, such as localized feelings of warmth, tingling, or an increase or
decrease in symptoms, within the period immediately following laser therapy. Other sensations that may be
experienced in response to laser therapy are nausea or dizziness. It is good practice to advice patients of this
possibility.
Treatment reactions, if they occur, are often reported after initial laser treatm ents, however, they generally diminish
after the second or third treatment. If a severe reaction is experienced during treatment, stop immediately.
To reiterate, optimal biostimulation is affected by the application of smaller dosages -per-point to more points at the
treatment site. Optimal bioinhibition is achieved through applying higher dosages-per-point, but to less treatment
points.
When treating acute musculoskeletal injuries, the initial desired outcome of laser therapy is the reduction of pain
and inflammation. It is very effective when used in conjunction with cryo-therapy, rest and elevation of the injury site.
Ideally treatment will begin as soon as possible after the injury occurs, with relatively high, inhibitory dosages (8 -12
Joules per- point, up to 10 points) being used to attenuate the pain and reduce the initial inflammatory response. A
treatment frequency of 1-2 sessions per day may be used for the first 2-4 days post-injury.
As the time post-injury progresses, dosages and treatment frequency may be reduced. In the period 5-10 days post-
injury, dosages of 6-8 Joules per point may be useful in promoting the rate of the inflammatory process and in
clearing its products from the injury site, thus allowing healing to begin sooner.
Moving into the healing phase, dosages are lowered and treatment frequency is reduced further. Throughout the
healing and rehabilitation phase of an injury, biostimulatory dosages (1 -4 Joules per point) are used to promote
tissue repair and- reduce scarring and adhesions. Higher doses may be used as required to alleviate any pain that
results from over-working the injured body part during rehabilitation.
When treating chronic injuries or pain, it is best to start with lower doses and then work up to the most effective dos e
for that particular patient, as a high initial dose may cause an unpleasant exacerbation of symptomatic pain.
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43. Page- 43-70
4.1 Biophysical Aspects & light transport theory
4.2 Laser - Tissue Interaction
4.3 The Mechanisms of Low Level Laser Therapy
4.4 Effects of Laser on Biological Cell/Tissue healing
Laser on hard tissue & Bone stimulation/ Regeneration
4.5 Medical application of Low Level Laser
4.1a- Biophysical Aspects of Low Level Laser Therapy (By Courtesy of-Herbert Klima, Atomic Institute of the Austrian
Universities, Vienna, Austria)
Biophysical aspects of low level laser therapy will be discussed from two points of view:
1. The electromagnetic and
2. The thermodynamical point of view.
From electromagnetic point of view,
Living systems are mainly governed by the electromagnetic interaction whose interacting particles are called photons. Each
interaction between molecules, macromolecules or living cells is basically electromagnetic and governed by photons. For
this reason, we must expect that electromagnetic influences like laser light of proper wavelength will have remarkable impact
on the regulation of living processes. An impressive example of this regulating function of various wavelengths of light is
found in the realm of botany, where photons of 660 nm are able to trigger the growth of plants which leads among other
things to the formation of buds. On the other hand, irradiation of plants by 730 nm photons may stop the growth and the
flowering. Human phagocyting cells are natively emitting light which can be detected by single photon counting methods.
Singlet oxygen molecules are the main sources of this light emitted at 480, 570, 633, 760, 1060 and 1270 nm- wavelengths.
On the other hand, human cells (leukocytes, lymphocytes, stem cells, fibroblasts, etc) can be stimulated by low power laser
light of just these wavelengths.
From thermodynamical point of view,
Living systems - in contrast to dead organisms - are open systems which need metabolism in order to maintain their highly
ordered state of life. Such states can only exist far from thermodynamical equilibrium thus dissipating heat in order to
maintain their high order and complexity. Such nonequilibrium systems are called dissipative structures proposed by the
Nobel laureat I. Prigogine. One of the main feature of dissipative structures is their ability to react very sensibly on weak
influences, e.g. they are able to amplify even very small stimuli.
Therefore, we must expect that even weak laser light of proper wavelength and proper irradiation should be able to influence
the dynamics of regulation in living systems. For example, the transition from a cell at rest to a dividing one will occur during
a phase transition already influenced by the tiniest fluctuations. External stimuli can induce these phase transitions which
would otherwise not even take place. These phase transitions induced by light can be impressively illustrated by various
chemical and- physiological reactions as special kinds of dissipative systems. One of the most important biochemical
reaction localized in mitochondria is the oxidation of NADH in the respiratory chain of aerobic cells.
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44. A similar reaction has been found to be a dissipative process showing oscillating and chaotic behavior capable to absorb
and amplify photons of proper wavelength.
A great variety of experimental and clinical results in the field of low level laser therapy supports these two biophysical
points of view concerning the interaction between life and laser light. Our former, but also our recent experimental results on
the effects of low level- laser light on human cells are steps in this direction.
By using cytometric, photometric and radiochemical methods it is shown that the increase or decrease of cells growth
depends on the applied wavelengths (480, 570, 633, 700, 760, 904, 1060, 1270 nm), on the irradiance (100 - 5000 J/m2),
on the pulse sequence modulated to laser beams (constant, periodic, chaotic pulses), on the type of cells- (leukocytes,
lymphocytes, fibroblasts, normal and cancer cells) and on the density of the cells in tissue cultures.
Our experimental results support our hypothesis which states that triplet oxygen molecules are able to absorb proper laser
light at wavelength at wavelengths 480, 570, 633, 700, 760, 904, 1060, 1270 nm thus producing singlet oxygen molecules.
Singlet oxygen takes part in many metabolic processes, e.g. catalytic oxidation of NADH which has been shown to be a
dissipative system far from thermodynamical equilibrium and sensitive even to small stimuli. Therefore, laser light of proper
wavelength and irradiance in low level laser therapy is assumed to be able- to excite oxygen molecules thus influencing or
amplifying metabolism and consequently influencing and supporting fundamental healing processes.
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45. 4.1b- Light transport theory
When light is sent into biological tissue, different processes can occur. While most of the light enters the tissue, a small part
of it can be reflected off the tissue surface. The amount of reflected light depends on the angle of incidence and the index of
refraction. Inside the tissue, the light can be absorbed or scattered. Both processes are highly wavelength dependent. In the
lower part of the visible wavelength region, the scattering probability is comparable to the absorption. In the red and near-
infrared wavelength region light penetrates tissue better. This region is called the optical window. Based on measurements
of optical properties, physiological or structural information about the probed tissue can be extracted. Here will outline the
mathematical basis of light transport in tissues and describe the tissue features that affect this transport.
The process of light transport in turbid media may be described mathematically either by analytical theory, based directly on
Maxwell's equations, or by transport theory. Maxwell's equations can describe the interaction between light and tissue as an
electromagnetic wave propagating through a medium with random dielectric fluctuations. However, due to the complex
structure of tissue it is in principle impossible to obtain a formulation that takes all its dielectric properties into account (A.
Ishimaru 1978). Transport theory, on the other hand, treats the problem as a flow of power through a scattering medium.
Transport theory is less mathematically- rigorous than electromagnetic theory and does not in itself include effects such as
diffraction or interference. However, it has proven useful for calculating photon transport in tissue. It is usually expressed for
the radiance L(r,s,t) [Wm-2sr-1], which is the radiant power per unit area and unit solid angle in direction s, at a position in
space r. It is obtained by multiplying the light distribution function, N(r,s,t) [m-3sr-1], with the speed and energy of the
photons in the medium. Radiance is the quantity used to describe the propagation of photon power. The transport equation
can be formulated as
Where c is the speed of light in the tissue, c = c0/n/ is the speed of light in vacuum and n is the refractive index of the
medium. The scattering and absorption coefficients μs and μa describe the probability of a scattering or absorption event
per unit length. The phase function, p(s, s‘) denotes the probability that a scattered photon initially travelling in direction s'
continues in direction s after the scattering event. The integral of the probability density function over all- solid angles dΩ' is
equal to one. The transport equation describes the energy balance in an infinitesimal volume element in the tissue.
The left-hand side of Equation (8) is the change in number of photons at position r, with direction s at time t. On the right-
hand side, the first two terms describe the loss of- photons, due to escape over the volume boundaries, scattering into other
directions or- absorption. The third term represents the gain through photons that are scattered from- other directions into
direction s, while the last term is gain due to a light source. It is assumed that all photons have the same energy and that all
scattering is elastic.
It is further assumed that the scattering is symmetric about the incident wave, which means that the phase function is a
function of the scattering angle alone, such that p(s,s')= p(θ), where θ is the angle- between the incident and the scattered
photon. It is often useful to have an analytic expression for the phase function.
The most popular phase function for light transport in biological tissue is the Henyey-Greenstein function(L.G Henyey
,J.LGreenstein 1941)
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46. Where g, the anisotropy factor, is the mean cosine of the scattering angle θ. For nearly isotropic scattering, the value of g is
close to zero, while a g close to unity indicates a strongly forward directed scattering. Tissue is in general highly forward
scattering. Analytic solutions to the transport equation are only known for a few special cases (K.M Case andP.F Zwiefel
1978). In practise, either numerical methods such as Monte Carlo simulations or expansion in spherical harmonics. The
Monte Carlo method simulates a migration of photon packages in a scattering and absorbing medium. The simulated
interaction events of these photon packages are based on random samplings from probability distributions of the step size
between interaction events and scattering angles. For each scattering event, the light is also attenuated due to absorption.
The trajectory of the photon package is followed until it exits through a boundary or is totally lost by absorption. The Monte
Carlo method is useful in that it can be used for any geometry, including layered and inhomogeneous media, and for any
optical properties. The main disadvantage is that, due to the statistical nature of the method, a large number of photon
packages have to be simulated, and requiring long computation times.
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47. 4.2 Laser - Tissue Interaction
Figure-4.2.1-Schematic diagram of laser-tissue interaction
Laser follows Lambert Beer law when it inter-acts with biological tissues.
Lambert Beer law
I = Io 10-aX
α= absorption coefficient
X = thickness of material/tissue
Io = incident intensity
I = transmitted intensity
Extinction length = 1/α = L; where 90% of the intensity is absorbed, when light energy is
reflected, transmitted, absorbed and scattered by interacting with biological tissues.
 Laser light can have the following effects with biological tissue:
1) Photochemical /Photodynamic effects
2) Photothermal effects
3) Mechanical effects
4) Photoablative effect.
 Photochemical
Laser energy can interact directly or indirectly with chemical structures within tissue. Photobiomodulation (laser
biostimulation, "cold laser" therapy): Low level laser or narrowband light has been used with varying success to modulate
cellular activity to achieve a biological effect such as stimulation of hair growth, collagen remodeling, accelerated wound
healing, etc. In most cases the mechanism of action remains unclear, although changes in mitochondrial activity or cell
membrane permeability may be responsible.
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