The Effect of Tension-Stress on the Genesis and Growth of Tissue
For a successful orthopaedic medicine physician, it is crucial to understand the mechanisms of connective tissue healing. While I will not cover all aspects of regenerative medicine, I would like to share a few thoughts with my colleagues.
Prolotherapy involves controlled injury of the enthesis. During injections, the extravasation of blood, mechanical injury, and osmotic effects of dextrose activate multiple physiological systems. These processes occur simultaneously in the tissues, working together to control and regulate complex physiological reactions. One such response to injury is the proliferation of fibroblasts, which transform into myofibroblasts. Myofibroblasts possess significant contractile abilities, generating tensile forces at the injection site, leading to elastic transformation.
Extensive medical, biological, and engineering studies have resulted in the discovery of a general biological law governing tissue growth and regeneration: The Law of Tension-Stress discovered by Gavriil Abramovich Ilizarov – Soviet Orthopedic Surgeon in 1954.
Gradual traction on living tissues creates stress that can stimulate and maintain the regeneration and growth of certain tissues. Slow, steady tension of tissue activates them metabolically, increasing their proliferative and biosynthetic functions. These processes depend on two main factors:
1. The quantity and quality of blood supply to the mechanically stressed tissue.
2. The stimulating effects of tensile forces along the lines of muscular contractions, as collagen fibers align parallel to the vector of tension-stress.
The clinical application of this biological law has allowed us to manipulate the healing process of soft tissue injuries, as well as certain diseases and disorders of the musculoskeletal system.
Numerous clinical and scientific observations confirm that tension-stress stimulates tissue growth, resembling the natural process of growth. Tension-stress stimulates osteogenesis and soft tissue histogenesis. The formation and growth of new tissue in adult organisms share similarities with tissue formation during embryonic and postnatal periods. For example, tension-stress induced by Myofibroblasts affects skeletal muscle, leading to changes in energy-supplying systems like mitochondria and protein-synthesizing systems like ribosomes and endoplasmic reticulum. Furthermore, tension-stress also stimulates the smooth muscle lining of blood vessels, increasing smooth muscle biosynthetic activity and proliferation, necessary for the healing of damaged ligaments and tendons.
Similar changes occur in connective tissue of fascia, tendons, dermis, as well as in the endomysium and perimysium of muscle, adventitia of blood vessels, and epineurium and perineurium of major nerve trunks.
Following an injury, the number of fibroblasts increases, and there is noticeable hypertrophy of the Golgi complex and enlargement of mitochondria, cytoskeletal microfilaments, and granular endoplasmic reticulum. These changes identify fibroblasts as type II collagenoblasts, which are typical of embryonic connective tissue. Tension-stress also stimulates elongation of nerve axons, eventually causing them to join one another.
These processes are not new; various healthcare practitioners utilize tension-stress in their practices. Major applications include orthopaedic surgery, prolotherapy, myofascial release, osteopathy, massage, and physical therapy. Combining different modalities will improve the success rate in managing patients with various musculoskeletal problems.
Jon Trister MD
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Inflamed tissue can become antigenic under certain circumstances. During inflammation, tissue damage and immune responses can lead to the release of self-antigens (molecules derived from the body’s own tissues) or modified self-antigens that may be recognized by the immune system. This process can occur in the following ways:
1. Release of Damage-Associated Molecular Patterns (DAMPs):
• Inflammation caused by injury or infection leads to the release of DAMPs from damaged cells. These molecules can act as signals to activate immune cells and may also serve as antigens that are presented to T cells, potentially triggering an immune response against self-tissues.
2. Antigen Presentation in Inflammatory Sites:
• During inflammation, antigen-presenting cells (APCs) like macrophages and dendritic cells are activated and can present antigens derived from inflamed or damaged tissues to T cells. This process can enhance the immune system’s recognition of these antigens.
3. Inflammation-Associated Self-Antigens:
• Some self-antigens are upregulated in inflamed tissues as part of the inflammatory response. These molecules may overlap spatially and temporally with foreign antigens, making it challenging for the immune system to distinguish between self and non-self. If tolerance mechanisms fail, these inflammation-associated self-antigens can trigger autoimmune responses.
4. Chronic Inflammation and Autoimmunity:
• Chronic inflammation increases the likelihood of self-antigen presentation and immune activation, which can lead to autoimmunity. For example, prolonged exposure of immune cells to tissue antigens in an inflammatory environment may result in a breakdown of tolerance and the development of diseases like rheumatoid arthritis or lupus.
In conclusion, inflamed tissue can indeed become antigenic when tissue damage or immune activation exposes or modifies self-antigens, potentially leading to immune responses against the body’s own tissues. This phenomenon is a key factor in the development of autoimmune diseases and chronic inflammatory conditions.
Damage-Associated Molecular Patterns (DAMPs) are implicated in a variety of diseases, particularly those involving inflammation and immune dysregulation. Below are examples of diseases associated with DAMPs:
Examples of DAMP-Associated Diseases
1. Autoimmune Diseases:
• Rheumatoid Arthritis (RA): DAMPs such as HMGB1 and S100 proteins contribute to joint inflammation and cartilage destruction by activating innate immune responses.
• Systemic Lupus Erythematosus : DAMPs like nuclear DNA and mitochondrial DNA are released during cell damage, exacerbating inflammation and promoting autoimmunity.
2. Cardiovascular Diseases:
• Atherosclerosis: DAMPs, including HMGB1 and S100 proteins, are involved in plaque formation and inflammation in arterial walls, contributing to the progression of atherosclerosis.
• Myocardial Infarction (MI): DAMPs released during ischemia-reperfusion injury, such as ATP and HMGB1, trigger sterile inflammation and tissue damage.
3. Neurodegenerative Diseases:
• Alzheimer’s Disease (AD): Elevated levels of HMGB1 and S100B in the brain contribute to neuroinflammation and blood-brain barrier dysfunction, worsening disease progression.
• Parkinson’s Disease: Similar mechanisms involving DAMP-induced neuroinflammation are observed in Parkinson’s.
4. Metabolic Diseases:
• Type 2 Diabetes Mellitus (T2DM): DAMPs generated by metabolic stress, such as ER stress-induced molecules, promote chronic inflammation that contributes to insulin resistance and tissue damage.
5. Infectious Diseases:
• Sepsis: High concentrations of DAMPs like nuclear DNA (nDNA), mitochondrial DNA (mtDNA), and heat shock proteins correlate with disease severity and poor outcomes in sepsis patients.
6. Cancer:
• Certain DAMPs, such as HMGB1, play dual roles in cancer by promoting tumor growth through inflammation or enhancing anti-tumor immunity under specific conditions.
7. Osteoarthritis (OA):
• Elevated levels of HMGB1 in synovial fluid and cartilage are associated with increased inflammation and cartilage destruction in OA patients.
DAMPs participate in the pathogenesis of many inflammatory, autoimmune, metabolic, neurodegenerative, cardiovascular, infectious, and degenerative diseases. Their involvement makes them potential biomarkers for diagnosis and targets for therapeutic intervention.
High Mobility Group Box 1 (HMGB1) is a highly conserved, non-histone nuclear protein that plays diverse roles depending on its location within or outside the cell. It is involved in DNA-related functions in the nucleus and acts as a damage-associated molecular pattern (DAMP) molecule when released extracellularly, triggering immune and inflammatory responses.
Functions of HMGB1
1. In the Nucleus:
• Acts as a DNA chaperone, maintaining chromosomal structure and regulating transcription, replication, DNA repair, and nucleosome assembly.
• Helps stabilize the genome and supports normal cellular processes.
2. In the Cytoplasm:
• Promotes autophagy by interacting with proteins such as BECN1 (Beclin-1), which is critical for cellular survival under stress conditions.
3. Extracellularly:
• Functions as a DAMP molecule, signaling tissue damage or danger.
• Activates immune responses by binding to receptors such as TLR4 (Toll-like receptor 4) and RAGE (Receptor for Advanced Glycation End-products).
• Triggers the production of pro-inflammatory cytokines and chemokines via pathways like NF-κB and MAP kinase signaling.
• Plays a role in recruiting immune cells to sites of injury or infection and amplifies inflammation.
Role in Diseases
HMGB1 is implicated in numerous pathological conditions due to its ability to sustain inflammation and immune activation:
• Neuroinflammatory Diseases: Involved in Parkinson’s disease, stroke, epilepsy, and multiple sclerosis by promoting neuroinflammation.
• Autoimmune Diseases: Contributes to diseases like rheumatoid arthritis and lupus by enhancing immune cell activation and autoantibody production.
• Cardiovascular Diseases: Plays a role in atherosclerosis and myocardial infarction through inflammatory pathways.
• Cancer: Can promote tumor growth or anti-tumor immunity depending on the context.
• Sepsis and Trauma: Acts as a key mediator of sterile inflammation in response to injury or infection.
Mechanisms of HMGB1 Release
• HMGB1 can be actively secreted by immune cells (e.g., macrophages, dendritic cells) during activation or passively released by necrotic or damaged cells.
• Its release is tightly regulated, and factors like oxidative stress or hypoxia can influence its secretion.
Therapeutic Potential
Targeting HMGB1 has shown promise in preclinical studies for various diseases:
• HMGB1 antagonists (e.g., glycyrrhizin) can reduce inflammation and tissue damage.
• Neutralizing antibodies against HMGB1 are being explored for autoimmune diseases, neuroinflammation , and sepsis.
In summary, HMGB1 is a multifunctional protein with critical roles in both homeostasis and disease. Its ability to act as an alarming makes it a key player in inflammatory processes, but it also presents opportunities for therapeutic intervention in conditions where excessive inflammation is detrimental.
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When an intramuscular injection of a circulating antigen, taken from venous blood, is administered, the immune response involves both the innate and adaptive immune systems. Here’s how each system responds:
Innate Immune Response
1. Local Inflammation: The injection site experiences an immediate local inflammatory response. This involves the recruitment of innate immune cells such as neutrophils, monocytes, and dendritic cells to the site of injection to initiate recognition and processing of the antigen.
2. Antigen Uptake and Presentation: Innate immune cells, particularly DCs, capture the antigen. These antigen-presenting cells (APCs) process the antigen and migrate to the draining lymph nodes. Here, they present the antigen (in the form of MHC-II with epitope ) to T cells, bridging the innate and adaptive immune responses.
3. Cytokine Production: The interaction with the antigen leads to the production of pro-inflammatory cytokines and chemokines, which further recruit immune cells to the site and enhance the inflammatory response.
Adaptive Immune Response
1. Activation of T Cells: Once in the lymph nodes, APCs present the processed antigens to CD4+ helper T cells via major histocompatibility complex (MHC) class II molecules. This activation is crucial for initiating a specific adaptive immune response.
2. B Cell Activation and Antibody Production: Helper T cells assist in activating B cells that recognize the same antigen. This leads to their differentiation into plasma cells that produce specific antibodies against the antigen.
3. Memory Formation: Some activated T and B cells become memory cells, providing long-term immunity by enabling a faster response upon future exposures to the same antigen.
Response to Antigen/Antibody Complexes
• Complex Formation: If antibodies are already present in circulation, they may bind to the injected antigens forming antigen-antibody complexes.
• Enhanced Phagocytosis: These complexes can enhance phagocytosis by binding to Fc receptors on phagocytes, leading to more efficient clearance of antigens.
• Complement Activation: The complexes can also activate the complement system, further promoting inflammation and opsonization.
Overall, while both innate and adaptive responses are activated by intramuscular injections of circulating antigens, the innate response provides immediate defense and sets up conditions for a more targeted adaptive response. The presence of pre-existing antibodies can modify this response by forming complexes that enhance certain immune pathways.
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The innate and adaptive immune systems respond differently to circulating antigens, tissue antigens, and antigen-antibody complexes due to their distinct mechanisms and roles in immunity.
Innate Immune System Response
• Circulating Antigens: The innate immune system provides an immediate, non-specific response. It uses pattern recognition receptors (PRRs) to identify common pathogen-associated molecular patterns (PAMPs) on circulating antigens. Key components include phagocytes (e.g., macrophages, neutrophils) that engulf pathogens, and the complement system, which enhances phagocytosis and can directly lyse pathogens.
• Tissue Antigens: When antigens are present in tissues, the innate immune system responds by recruiting immune cells to the site of infection or injury. This involves the release of cytokines and chemokines that promote inflammation and attract cells like neutrophils and macrophages to the affected area.
• Antigen-Antibody Complexes: These complexes can activate the complement system, leading to opsonization and enhanced phagocytosis by innate immune cells. Fc receptors on innate cells bind to antibodies in the complexes, triggering phagocytosis or cell activation.
Adaptive Immune System Response
• Circulating Antigens: The adaptive immune response is slower but more specific. Circulating antigens are typically captured by antigen-presenting cells (APCs) like dendritic cells, which process and present them to T cells in lymphoid tissues. This initiates a specific T cell response, leading to clonal expansion and differentiation into effector T cells.
• Tissue Antigens: Adaptive responses to tissue antigens involve effector T cells migrating to the site of infection or inflammation. CD8+ cytotoxic T cells can directly kill infected cells, while CD4+ helper T cells assist by activating other immune cells such as macrophages.
• Antigen-Antibody Complexes: These complexes can also influence adaptive immunity by enhancing antigen presentation. B cells can bind these complexes through their B-cell receptors (BCRs), leading to activation and differentiation into antibody-secreting plasma cells. Additionally, immune complexes can modulate antibody production through feedback mechanisms involving Fc receptors on B cells.
Synergy Between Innate and Adaptive Responses
The innate immune system often acts as a first line of defense, providing immediate responses that help contain infections until the adaptive immune system is activated. Information from the innate response is responsible for activating adaptive immunity; for example, cytokines produced by innate immune cells influence the type of adaptive response generated. Adaptive responses then refine and amplify the initial defense provided by innate immunity, often involving memory formation for quicker responses upon re-exposure to the same antigen.
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Vitamin C and B vitamins play distinct roles in the prevention of cardiovascular disease , primarily through their antioxidant, anti-inflammatory, and metabolic effects. Here’s an overview of their contributions:
Vitamin C and Cardiovascular Disease Prevention
Vitamin C (ascorbic acid) is a powerful antioxidant that may help reduce the risk of CVD through several mechanisms:
1. Antioxidant Effects: Vitamin C protects low-density lipoprotein cholesterol from oxidative damage, which is a key step in the development of atherosclerosis. It also reduces oxidative stress by scavenging free radicals.
2. Improved Endothelial Function: Vitamin C enhances nitric oxide bioavailability, promoting vasodilation and improving blood flow. This helps reduce blood pressure and supports arterial health.
3. Reduction in Inflammation: By inhibiting the adhesion of monocytes to endothelial cells, vitamin C may prevent early stages of atherosclerosis. It also stabilizes atheromatous plaques, reducing the risk of rupture.
4. Blood Pressure Regulation: Studies suggest that vitamin C supplementation can lower both systolic and diastolic blood pressure in hypertensive individuals, which is beneficial for heart health.
5. Mixed Evidence on Mortality: While observational studies have linked higher vitamin C intake to lower CVD mortality, randomized controlled trials (RCTs) have shown inconsistent results regarding its ability to prevent cardiovascular events or death.
B Vitamins and Cardiovascular Disease Prevention
B vitamins, particularly folic acid (B9), vitamin B6, and vitamin B12, are essential in cardiovascular health due to their role in homocysteine metabolism:
1. Homocysteine Reduction: Elevated homocysteine levels are an independent risk factor for atherosclerosis and other cardiovascular conditions. B vitamins lower homocysteine levels, potentially reducing the risk of CVD.
2. Carotid Intima-Media Thickness (CIMT): Folic acid supplementation has been shown to reduce CIMT, a marker of atherosclerosis progression.
3. Anti-Inflammatory Effects: Vitamin B6 may exert cardiovascular benefits by reducing inflammation through pathways such as the kynurenine pathway.
4. Dietary Intake and Risk Reduction:
• Higher dietary intake of vitamin B6 has been associated with a lower prevalence of CVD in population studies.
• Folic acid and vitamin B12 supplementation have shown benefits in reducing stroke risk but not consistently for other cardiovascular outcomes.
5. Limitations: While some studies suggest benefits from B vitamin supplementation, others report no significant reduction in cardiovascular events or mortality. Confounding factors such as concurrent use of statins or aspirin may obscure results.
Summary
• Vitamin C: Its antioxidant properties and effects on endothelial function suggest it may help prevent early stages of atherosclerosis, lower blood pressure, and support overall vascular health. However, evidence from RCTs on its ability to prevent major cardiovascular events remains inconclusive.
• B Vitamins: By lowering homocysteine levels and reducing inflammation, folic acid, vitamin B6, and vitamin B12 may contribute to cardiovascular health. Their greatest benefits appear to be in specific populations with high homocysteine levels or normal renal function.
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Inflamed tissue can become antigenic under certain circumstances. During inflammation, tissue damage and immune responses can lead to the release of self-antigens (molecules derived from the body’s own tissues) or modified self-antigens that may be recognized by the immune system. This process can occur in the following ways:
1. Release of Damage-Associated Molecular Patterns (DAMPs):
• Inflammation caused by injury or infection leads to the release of DAMPs from damaged cells. These molecules can act as signals to activate immune cells and may also serve as antigens that are presented to T cells, potentially triggering an immune response against self-tissues.
2. Antigen Presentation in Inflammatory Sites:
• During inflammation, antigen-presenting cells (APCs) like macrophages and dendritic cells are activated and can present antigens derived from inflamed or damaged tissues to T cells. This process can enhance the immune system’s recognition of these antigens.
3. Inflammation-Associated Self-Antigens:
• Some self-antigens are upregulated in inflamed tissues as part of the inflammatory response. These molecules may overlap spatially and temporally with foreign antigens, making it challenging for the immune system to distinguish between self and non-self. If tolerance mechanisms fail, these inflammation-associated self-antigens can trigger autoimmune responses.
4. Chronic Inflammation and Autoimmunity:
• Chronic inflammation increases the likelihood of self-antigen presentation and immune activation, which can lead to autoimmunity. For example, prolonged exposure of immune cells to tissue antigens in an inflammatory environment may result in a breakdown of tolerance and the development of diseases like rheumatoid arthritis or lupus.
In conclusion, inflamed tissue can indeed become antigenic when tissue damage or immune activation exposes or modifies self-antigens, potentially leading to immune responses against the body’s own tissues. This phenomenon is a key factor in the development of autoimmune diseases and chronic inflammatory conditions.
Damage-Associated Molecular Patterns (DAMPs) are implicated in a variety of diseases, particularly those involving inflammation and immune dysregulation. Below are examples of diseases associated with DAMPs:
Examples of DAMP-Associated Diseases
1. Autoimmune Diseases:
• Rheumatoid Arthritis (RA): DAMPs such as HMGB1 and S100 proteins contribute to joint inflammation and cartilage destruction by activating innate immune responses.
• Systemic Lupus Erythematosus : DAMPs like nuclear DNA and mitochondrial DNA are released during cell damage, exacerbating inflammation and promoting autoimmunity.
2. Cardiovascular Diseases:
• Atherosclerosis: DAMPs, including HMGB1 and S100 proteins, are involved in plaque formation and inflammation in arterial walls, contributing to the progression of atherosclerosis.
• Myocardial Infarction (MI): DAMPs released during ischemia-reperfusion injury, such as ATP and HMGB1, trigger sterile inflammation and tissue damage.
3. Neurodegenerative Diseases:
• Alzheimer’s Disease (AD): Elevated levels of HMGB1 and S100B in the brain contribute to neuroinflammation and blood-brain barrier dysfunction, worsening disease progression.
• Parkinson’s Disease: Similar mechanisms involving DAMP-induced neuroinflammation are observed in Parkinson’s.
4. Metabolic Diseases:
• Type 2 Diabetes Mellitus (T2DM): DAMPs generated by metabolic stress, such as ER stress-induced molecules, promote chronic inflammation that contributes to insulin resistance and tissue damage.
5. Infectious Diseases:
• Sepsis: High concentrations of DAMPs like nuclear DNA (nDNA), mitochondrial DNA (mtDNA), and heat shock proteins correlate with disease severity and poor outcomes in sepsis patients.
6. Cancer:
• Certain DAMPs, such as HMGB1, play dual roles in cancer by promoting tumor growth through inflammation or enhancing anti-tumor immunity under specific conditions.
7. Osteoarthritis (OA):
• Elevated levels of HMGB1 in synovial fluid and cartilage are associated with increased inflammation and cartilage destruction in OA patients.
DAMPs participate in the pathogenesis of many inflammatory, autoimmune, metabolic, neurodegenerative, cardiovascular, infectious, and degenerative diseases. Their involvement makes them potential biomarkers for diagnosis and targets for therapeutic intervention.
High Mobility Group Box 1 (HMGB1) is a highly conserved, non-histone nuclear protein that plays diverse roles depending on its location within or outside the cell. It is involved in DNA-related functions in the nucleus and acts as a damage-associated molecular pattern (DAMP) molecule when released extracellularly, triggering immune and inflammatory responses.
Functions of HMGB1
1. In the Nucleus:
• Acts as a DNA chaperone, maintaining chromosomal structure and regulating transcription, replication, DNA repair, and nucleosome assembly.
• Helps stabilize the genome and supports normal cellular processes.
2. In the Cytoplasm:
• Promotes autophagy by interacting with proteins such as BECN1 (Beclin-1), which is critical for cellular survival under stress conditions.
3. Extracellularly:
• Functions as a DAMP molecule, signaling tissue damage or danger.
• Activates immune responses by binding to receptors such as TLR4 (Toll-like receptor 4) and RAGE (Receptor for Advanced Glycation End-products).
• Triggers the production of pro-inflammatory cytokines and chemokines via pathways like NF-κB and MAP kinase signaling.
• Plays a role in recruiting immune cells to sites of injury or infection and amplifies inflammation.
Role in Diseases
HMGB1 is implicated in numerous pathological conditions due to its ability to sustain inflammation and immune activation:
• Neuroinflammatory Diseases: Involved in Parkinson’s disease, stroke, epilepsy, and multiple sclerosis by promoting neuroinflammation.
• Autoimmune Diseases: Contributes to diseases like rheumatoid arthritis and lupus by enhancing immune cell activation and autoantibody production.
• Cardiovascular Diseases: Plays a role in atherosclerosis and myocardial infarction through inflammatory pathways.
• Cancer: Can promote tumor growth or anti-tumor immunity depending on the context.
• Sepsis and Trauma: Acts as a key mediator of sterile inflammation in response to injury or infection.
Mechanisms of HMGB1 Release
• HMGB1 can be actively secreted by immune cells (e.g., macrophages, dendritic cells) during activation or passively released by necrotic or damaged cells.
• Its release is tightly regulated, and factors like oxidative stress or hypoxia can influence its secretion.
Therapeutic Potential
Targeting HMGB1 has shown promise in preclinical studies for various diseases:
• HMGB1 antagonists (e.g., glycyrrhizin) can reduce inflammation and tissue damage.
• Neutralizing antibodies against HMGB1 are being explored for autoimmune diseases, neuroinflammation, and sepsis.
In summary, HMGB1 is a multifunctional protein with critical roles in both homeostasis and disease. Its ability to act as an alarmin makes it a key player in inflammatory processes, but it also presents opportunities for therapeutic intervention in conditions where excessive inflammation is detrimental.
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In 1977, an article detailing the mechanisms of osteogenesis was published in the scientific journals of Ilizarov’s institute. Many of the concepts discussed in this article are still relevant to our current understanding of tissue repair following an injury. Below is a translation of the article from Russian.
UDC 611-018.4+616.71-003.93
A. M. KHELIMSKIY.
OSTEOGENIC COLONY FORMATION IN REPARATIVE REGENERATION OF BONE TISSUE
The phenomenon of bone tissue formation during cultivation of osteogenic cells (Friedenstein A. Ya., Lalykina K. S., 1973), discovered in recent years (see the article by R. Ya. Galanova et al. in this collection), served as the basis for us to interpret the pictures of reparative bone formation in a new way. This concept of osteogenesis is presented below.
Bone damage is accompanied by two processes: 1) blood containing a certain amount of colony-forming osteogenic cells constantly circulating in the blood flows into the injury zone; 2) distant and local regulators are formed in the injury zone. Let us conventionally call the first of them a pheromone (from the Latin fero to carry, tonco to persuade), the second an attractant (from the Latin attraho to attract). The pheromone, spreading through the blood or lymph through veins or lymphatic pathways, forces the precursor cells of osteogenesis to leave the bone marrow of various bones of the body into the general blood flow. The attractant remains in the area of injury and attracts colony-forming cells of osteogenesis, giving them a signal to begin column formation. Therefore, bone formation occurs precisely at the site of injury, despite the circulation of precursor cells in the general blood flow. Both the pheromone and the attractant (regardless of their structure, mono- or polycomponent nature, etc.) must first of all be products of bone tissue disintegration, since if they were of a different origin, for example, a product of the destruction of the vascular wall, blood, fibrous tissue, etc., they would not be specific to bone formation and would not attract specifically the stem cells of osteogenesis to this focus.
Thus, if, for example, we inflict a repeated injury some time after bone injury, healing will proceed faster, since the attractant in the new place will attract pre-stimulated pheromone-stimulated cells – precursors of osteogenesis. It is only necessary that the interval between the first and repeated injuries correspond to a certain “incubation period”. We believe that the time of the onset of distraction should be partly dictated by the size of this period in each specific case.
The pheromone, of course, activates column-forming cells not only in distant parts of the body, but also in the bone marrow of the damaged bone. The attractant draws them to the injury zone. The process of osteogenic column formation, the development of a clone of bone-forming cells, begins. Three stages of colony formation can be outlined: Stage 1: formation of colonies from colony-forming cells of the hematoma in places where there are suitable conditions for this; Stage 2: formation of osteogenic colonies from activated local precursor cells in the bone marrow of the damaged bone (the beginning of the endosteal reaction). At this time, there are many small “foci” (osteogenesis colonies) in the regeneration zone. What is a colony during bone formation?. This is the offspring of one stem cell, gradually passing through the stages of differentiation through fibroblasts, preosteoblasts, osteoblasts to osteocytes, and the products of their excretory function, forming the bone matrix. The center of the column is its more mature, more differentiated sections. On the periphery are younger individuals. In the bone marrow, under conditions of stable fixation, the colonies are rounded, on cortical plates or large bone fragments they are spread out in the form of plaques (the more mature part of the colony on the base of the plaque). Often the center of the colony is located near the bone fragment of the attractant localization. Stage 3: formation of colonies from colony-forming cells that have arrived through the blood and penetrate into the damage zone along the supplying bone vessels of the periosteum or endosteum. Perosteal colonies arise in places of preserved blood supply; due to mechanical reasons they also spread out on the cortical plate in the form of plaques. The endosteal regenerate is now composed of three sources: colonies that arose in the hematoma, colonies from local precursor cells; colonies from alien precursor cells of osteogenesis. All these colonies merge as they develop, the distal part of the regenerate (where colony formation usually lags behind due to blood flow conditions) merges with the proximal part. By about day 14 after osteotomy, the colony “matures”, turning into a bone focus surrounded by active osteoblasts.The diameter of the focus is 1-2 mm. The further fate of the formed bone depends on biomechanical conditions: either it will develop as a result of the activity of osteoblasts, or in the absence of load, osteocytes will begin to die, and osteoclasts will resorb the decaying bone.
Although the center of bone formation is the vessel supplier of colony-forming cells, as the colony develops, its center, where bone formation begins, seems to move away from the vessel. As the colony matures, proliferation ceases and bone is replaced, the bone again “approaches” the vessel, now directly adjacent to it. Thus, the type of osteogenesis (desmal or angiogenic) is more appropriately considered as a manifestation of the “packing density” of the vessels, and, consequently, osteogenic colonies.
With a lack of nutrition and energy, osteogenesis is distorted, the colony turns into a chondroid focus.
It is obvious that the activity of the reparative reaction depends on the volumetric level of blood flow in a given area. The more intense the blood supply, the more intensive the delivery of colony-forming cells, nutrition and energy for the colonies. Therefore, metaphyseal injuries heal faster than diaphyseal injuries. The Ilizarov method, which preserves muscle and joint function, i.e. ensures blood and lymph flow, and the
Thus, the transport of pheromone, as well as ensuring the integrity of vessels and osteogenesis colonies in the contact zone (due to stable fixation of fragments), creates optimal conditions for bone regeneration. Bone formation should be considered from this standpoint during distraction. Distraction constantly maintains the release of pheromone and attractant, i.e. constantly maintains the influx of column-forming cells into the stretch zone. The middle growth zone in the distraction regenerate is abundantly supplied with vessels. As the density increases, it moves further and further away from the ends of the fragments. Newly formed osteoid beams in the growth zone are most exposed to the action of stretching, i.e. the formation of beams is combined with their partial disintegration, disintegration of the bone matrix. The attractant is localized here, and the vessels deliver colony-forming cells here. Here the “packing density” of the vessels is high enough to form merging lace-like colonies of bone formation around them, i.e. for the manifestation of the so-called direct angiogenic osteogenesis. At the same time, endosteal and perosteal bone formation in the fragments increasingly subsides. As the experiments of A. A. Shreiner showed, an abundant vascular network develops during distraction. At first, it is located at the ends of the fragments, corresponding to the development of endosteal foci of osteogenesis. As distraction progresses, the zone of abundant blood supply shifts toward the center of the regenerate, toward its growth zone. V. G. Berko’s studies have shown that in the growth zone the number of vessels during distraction increases on average by 1 vessel per day per 1 sq. mm of section area (during femur lengthening in dogs). Accordingly, the space between the vessels decreases. The area of the functional-structural unit of bone formation decreases during distraction from 0.040 to 0.015 mm². From the 21st to 28th day of distraction, the picture corresponding to desmal osteogenesis is replaced by a picture of direct
angiogenic bone formation. These data can be compared with the observations of
K. V. Petrakova, A. A. Tolmacheva, A. Ya. Friedenstein (1963), who showed that the direction of differentiation of the osteogenic cell culture depends on the “packing density”. Thus, when growing the same number of osteogenic cells in vivo in a chamber with an area of 50 mm², reticular tissue was formed, and in a chamber with an area of 5 mm², foci of bone formation appeared.
After distraction, during the period of subsequent fixation, the stretching of the beams ceases, the production of the Pheromone and attractant ceases, the colonies mature and the growth zone is replaced by bone.
If stability is not ensured during the healing of a bone wound, the level of injury is high, there are many colonies, but they are partially destroyed, and excessive but disordered bone formation occurs.
In the places where the spokes are inserted, the pattern of bone formation is naturally similar to that observed in other areas of damage. But if the spoke is under the action of compression or distraction forces, colony formation is induced in a certain direction. Probably, such pathological processes as ossifying myositis, ossification of tendons after injury, etc., are also the result of accidental formation of an attractant in an unusual place and the occurrence of osteogenic colony formation.
As for physiological regeneration and bone remodeling, it seems to us that this is the result of activation or inactivation under the influence of overload or inactivity of osteoblasts remaining on the bone surface (endosteum, periosteum, osteon canals), and not the result of attracting circulating colony-forming cells.
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Prolotherapy is a controlled injury utilizing mechanical, osmotic, chemical stimuli.As the result Activation of Mesenchymal Stem Cells (MSCs) occurs.
Injury Signals:
When tissue sustains damage, it releases a variety of signals, including cytokines, chemokines, and growth factors. These signals serve to attract MSCs to the site of injury and activate their functions.
MSCs from surrounding tissues and the bloodstream migrate to the wound site, drawn by chemotactic factors that are released during the initial inflammatory response.
Initiation of the Healing Process:
1. Hemostasis and Inflammation:
MSCs secreting pro-inflammatory cytokines, which help recruit neutrophils and M1 macrophages to clear debris and combat potential infections.
2. Immunomodulation:
MSCs begin to modulate the immune response, decreasing the levels of activated T cells, neutrophils, and macrophages. They also guide the polarization of monocytes into pro-reparative M2 macrophages.
3. Paracrine Signaling:
MSCs release a range of growth factors and cytokines that facilitate healing. These include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and transforming growth factor-β (TGF-β).
Control of the Healing Process:
1. Angiogenesis:
MSCs promote the formation of new blood vessels by secreting pro-angiogenic factors.
2. Cell Proliferation and Migration:
MSCs stimulate the proliferation and migration of skin cells, such as fibroblasts and keratinocytes.
3. Extracellular Matrix (ECM) Regulation:
MSCs participate in production and remodeling of the ECM by releasing matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). They also promote collagen and elastin production by fibroblasts.
4. Re-epithelialization:
MSCs enhance the migration and proliferation of keratinocytes, which is essential for wound closure.
5. Granulation Tissue Formation:
MSCs contribute to the development of granulation tissue, characterized by a rich collagen III matrix.
6. Anti-fibrotic Activity:
MSCs help prevent excessive scarring by maintaining a balance between TGF-β1 and TGF-β3.
7. Resolution of Inflammation:
As healing progresses, MSCs continue to modulate the inflammatory response, facilitating a transition from pro-inflammatory to anti-inflammatory conditions.
Throughout the healing process, MSCs demonstrate plasticity, adapting their functions to the changing needs of the wound environment. They orchestrate the complex interplay between various cell types and signaling molecules, promoting a regenerative rather than a fibrotic healing response
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Connective tissue healing. Review literature by Jon Trister MD
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Connective tissue healing s a systematic process that can be categorized into four overlapping stages: hemostasis, inflammation, proliferation, and remodeling. Each stage is marked by distinct cellular activities and accompanying symptoms.
1. Hemostasis (Immediate to 1-2 days)
This stage serves as the body’s immediate response to injury, aimed at halting bleeding.
Key processes: Blood vessels constrict , and platelets aggregate to form a blood clot that seals the wound. This clot acts as a temporary matrix for the healing process.
Symptoms: While minimal symptoms may be present, slight swelling or redness at the injury site may occur as clotting commences.
Key Cells: Platelets, the principal cells in this phase, are small, disc-shaped fragments that adhere to damaged endothelium, releasing factors like fibrinogen and von Willebrand factor to form a clot (thrombus). Their primary function is to prevent further bleeding and provide a scaffold for subsequent healing stages.
Mesenchymal stem cells help by secreting factors that modulate the inflammatory response. They release pro-inflammatory cytokines that recruit immune cells like neutrophils and macrophages to the wound site, which are essential for clearing debris and preventing infection. MSCs also help form a stable clot by interacting with platelets and fibrin mesh.
2. Inflammation (1-7 days) Inflammation acts as the body’s defense mechanism, cleaning the wound and preparing for tissue repair.
Key processes: White blood cells, such as neutrophils and macrophages, migrate to the injury site to eliminate debris and bacteria. This phase also sees vasodilation, which enhances blood flow to the area.
Symptoms: Expect swelling, redness, warmth, pain, and occasionally a loss of function. These symptoms arise from increased blood flow and immune responses in the affected area.
Key Cells:Neutrophils:Multi-lobed, granular white blood cells that arrive at the injury site within hours, engulfing pathogens and debris through phagocytosis.
Macrophages: Large, amoeboid cells with kidney-shaped nuclei that arrive later; they phagocytose dead cells and release cytokines to attract more immune cells and encourage tissue repair.
Mast Cells: Containing granules rich in histamine, these cells promote vasodilation and increase vascular permeability, facilitating immune cell influx.
Mesenchymal stem cells exert immunomodulatory effects. They reduce excessive inflammation by shifting macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type. This polarization helps clear apoptotic cells and debris while promoting tissue repair. MSCs also secrete anti-inflammatory cytokines like IL-10, which further dampen the inflammatory response.
3.Proliferation (3 days to 3 weeks)
This stage focuses on rebuilding damaged tissue.
Key processes: Fibroblasts generate collagen and extracellular matrix, forming granulation tissue. Angiogenesis occurs as new blood vessels form, and myofibroblasts contract the wound, pulling its edges together.
Symptoms: The injury site may appear pink or red due to new blood vessel formation. It may feel tender but less painful than during inflammation, and some itching may be experienced as new tissue develops.
Key Cells: Fibroblasts, myofibroblasts, endothelial cells, and keratinocytes.
Morphology:
Fibroblasts: Spindle-shaped cells synthesizing collagen and extracellular matrix (ECM). They become activated during this phase, exhibiting enlarged nuclei and abundant rough endoplasmic reticulum for ECM production.
Myofibroblasts: Derived from fibroblasts, these cells possess characteristics of both fibroblasts and smooth muscle cells, including actin filaments that aid in wound contraction.
Endothelial Cells: Flat, elongated cells that proliferate to form new blood vessels (angiogenesis), ensuring a sufficient oxygen supply to healing tissue.
Keratinocytes: Epithelial cells that migrate from the wound edges to re-establish the epidermis, appearing flattened as they spread across the wound bed.
Mesenchymal stem cells are involved in tissue regeneration. They promote the proliferation and migration of fibroblasts, keratinocytes, and endothelial cells, which are essential for angiogenesis, collagen deposition, and re-epithelialization of the wound. MSCs also stimulate granulation tissue formation, which is important for filling the wound bed with new extracellular matrix .
4. Remodeling (3 weeks to 1 year or more)
These stage involves strengthening and reorganizing newly formed tissue.
Key processes:Collagen fibers transition from disorganized type III collagen to the stronger type I collagen. The tissue gradually regains strength, although it will never be as strong as uninjured tissue.
Symptoms: There is typically reduced swelling and pain, but the skin or connective tissue may feel tight or stiff as scar tissue develops. Over time, scars may become less noticeable but will remain weaker than the original tissue.
Key Cells: Myofibroblasts, fibroblasts, and apoptotic immune cells.
Morphology:
Myofibroblasts: Continue to contract the wound while depositing type I collagen. Their actin filaments are prominent in this stage, enhancing ECM strength.
Fibroblasts: Remodel the collagen matrix by converting type III collagen to stronger type I collagen. They become less active as remodeling progresses, but are crucial for maintaining tissue integrity.
Many immune cells undergo apoptosis (programmed cell death) once their role in healing is complete, resulting in decreased cellularity at the injury site.
Mesenchymal stem cells contribute to tissue remodeling by regulating ECM turnover through the secretion of matrix metalloproteinases and their inhibitors. This process helps replace disorganized collagen III with stronger collagen I, reducing scarring and improving tissue strength. Additionally, MSCs help prevent fibrosis by maintaining a balance between different growth factors like TGF-β1 and TGF-β3.
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involves both intracellular and extracellular stages. It is essential for the formation of collagen, the most abundant protein in the body, which provides structural support to various tissues, including skin, bones, tendons, and cartilage. Here are the key steps involved in collagen synthesis:
- Transcription and Translation
• Transcription: The process begins inside the cell with the transcription of collagen genes into messenger RNA (mRNA). This occurs in the nucleus of fibroblasts (specialized cells responsible for collagen production).
• Translation: The mRNA is transported to ribosomes on the rough endoplasmic reticulum , where it is translated into a polypeptide chain known as preprocollagen. - Hydroxylation
• Hydroxylation of Proline and Lysine: The preprocollagen undergoes post-translational modifications in the RER. Specifically, certain proline and lysine residues are hydroxylated by enzymes (prolyl and lysyl hydroxylases) to form hydroxyproline and hydroxylysine. This step requires vitamin C as a cofactor.
• This hydroxylation is crucial for stabilizing the collagen triple helix structure. - Glycosylation
• Glycosylation of Hydroxylysine: After hydroxylation, some hydroxylysine residues are glycosylated with glucose or galactose sugars. This modification helps in the proper folding and assembly of procollagen. - Formation of Procollagen
• Triple Helix Formation: Three alpha chains (two alpha-1 chains and one alpha-2 chain) twist together into a triple helix structure, forming procollagen. This helical structure is stabilized by hydrogen and disulfide bonds. - Transport to Golgi Apparatus
• The procollagen molecules are transported from the RER to the Golgi apparatus, where they undergo further modifications and are packaged into vesicles for secretion. - Exocytosis
• Secretion into Extracellular Space: Procollagen is secreted from the cell through exocytosis into the extracellular matrix (ECM). - Cleavage of Propeptides
• Formation of Tropocollagen: Once outside the cell, specific enzymes called collagen peptidases cleave off the terminal propeptides from procollagen, converting it into tropocollagen, which is now capable of forming fibrils. - Cross-Linking and Fibril Formation • Cross-Linking by Lysyl Oxidase: Tropocollagen molecules spontaneously assemble into collagen fibrils. The enzyme lysyl oxidase (which requires copper as a cofactor) catalyzes covalent cross-linking between lysine residues, strengthening these fibrils. • These fibrils then aggregate to form larger collagen fibers, which provide tensile strength to tissues like skin, tendons, and bones. Summary of Key Steps
- Transcription and translation of preprocollagen.
- Hydroxylation of proline and lysine residues (vitamin C-dependent).
- Glycosylation of hydroxylysine residues.
- Formation of procollagen triple helix.
- Transport through Golgi apparatus.
- Secretion into extracellular space.
- Cleavage of propeptides to form tropocollagen.
- Cross-linking by lysyl oxidase to form collagen fibrils.
This intricate process ensures that collagen maintains its structural integrity, but disruptions at any step can lead to disorders such as scurvy (vitamin C deficiency), Ehlers-Danlos syndrome (defective cross-linking), or osteogenesis imperfecta (defective triple helix formation).
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