Table of Contents

Introduction

Innovative Equine Regenerative Therapies are the need of the hour to save horses from getting overloaded with pharmaceutical agents, i.e. drugs. There are many reasons to adopt such therapies. It is actually the ethical responsibility of clinicians to not only treat patients with strong dosages of drugs, but to find innovative solutions which are less harmful and more safe for the whole body. Many times, due to a lack of availability of the latest therapies, patients couldn’t be saved/kept alive. This is of great concern.

The good news is that there are many new Equine Regenerative Therapies available presently and more are in clinical trials, which means they will also be available soon. The purpose to adopt these therapies is to help patients avoid great and prolonged pain as well as treat them in a safer way. Life is precious, we must strive hard to protect it.

You will find more information about some of the advanced Equine Regenerative Therapies which are being used these days in this booklet. Some examples of this include: ozone, prolozone, equine hyperbaric oxygen therapy, stem cell therapy, platelet rich plasma (PRP), autologous conditioned serum (IRAP), and amniotic products.

Equine Ozone Therapy

Introduction

The delivery of oxygen and its availability, glucose, and ATP in the ischemic tissues is increased with the body tissues after ozone therapy. Bone marrow implantation can be enhanced at the lesion site increasing angiogenesis, neovascularisation, and tissue regeneration. The neurohumoral reaction can be activated so that it can improve the quality of life. The expression of antioxidant enzymes and heme-oxygenase can be upregulated producing the preconditioning benefits.

How It Works

A regular oxygen molecule, when treated with high energy, can be spliced into two single oxygen atoms. The single oxygen atom binds with a regular oxygen molecule to form ozone. The extra oxygen atom then becomes a scavenger, destroying viruses, bacteria, pollutants, and odors. The third oxygen atom has a half-life of 20 minutes and it is very powerful in contributing the destruction of viruses, fungi, and bacteria. There are several methods of administration of ozone to horses (Sciorsci, Lillo, Occhiogrosso, & Rizzo, 2020).

Auto-hemotherapy

The major autohemotherapy includes the collection of 250ml of blood from the animal in heparin or 3.13% sodium citrate anticoagulant and blood ozonation is done out of the body for 5-10 minutes. The ozonated blood is infused slowly back to the animal body through intravenous routes. This autohemotherapy approach was used in treating chronic laminitis and mechanical lumbar pain in a 10-year-old mare and riding horses respectively (Coelho et al., 2015).

Ozone Bagging:

This method involves the use of ozone resistant bag pumped with the ozone oxygen mixture and then it is placed near the area to be treated. Superficial lesions treatments can be done because of ozone absorption into the skin. Cutaneous infections like chronic wounds and ulcers are being treated with this method.

Ozonated oil:

Oil can be used as a carrier of Ozone. Ozone bubbling is done in the oil like olive, sesame, or sunflower. Upon forming the gel consistency, it can be used for the treatment of infections like skin wounds, insect stings, ulcers, vulvovaginitis, and periodontitis.

Ozony Blanket:

The whole body of an animal is ozonated by using an ozonated silicone blanket being placed around the horse’s body. This system is an effective way of treating various equine diseases.

Clinical Application of Ozone Therapy in Equine

Clinical studies show that ozone therapy is useful to treat several equine complications including arthritis, muscular pain and spasms, viral and bacterial infections, immunosuppressive conditions, and neurologic syndromes associated with hypoxia and infectious agents (J. Bhatt et al., 2016).

A combinatorial therapy involving PRP, ozone, and rehabilitation was employed to treat equine lameness. It showed some exciting results (figure 3) that are very encouraging to continue using this approach.

Another combinatorial therapy involving PRP and ozone therapy was used to treat OCD in horses. It yields impressive outcomes (figure 4) that present evidence of the efficiency of this treatment.

Prolozone Therapy

It is an injection-based, non-surgical, treatment to treat several chronic and degenerative diseases by facilitating the body’s own natural ability to recover. The word prolozone is a combination of two words, ‘proliferation’ and ‘ozone’. The prolozone therapy is stimulated by ozone and other nutrients to trigger healing, regeneration, and regrowth within the weakened area of the body. This therapy involves an injection with various combination of procaine, anti-inflammatory medications, minerals and vitamins, and ozone to the affected area of the body.

Ligaments are the structures that hold together the joints, bones, and intervertebral discs. They can be affected as a result of injuries. The weakening of ligaments could cause pain and arthritis in the bones, joints, and discs.

This therapy works almost similar to the ozone therapy involving an injection of ozone into and around the area where ligaments and the bones are attached together. This ozone injection will boost the supply of blood, flow of minerals, and nutrients to the affected area and recruitment of fibroblasts. So, an increase in the flow of nutrients and the availability of fibroblasts makes it an ideal therapy for arthritis. Procaine acts to re-establish cellular membrane potentials. Anti-inflammatory agents decrease edema and swelling. Vitamins and minerals provide necessary substrates for oxygen utilization to overcome oxygen deficiency in damaged tissues. This oxygen utilization is stimulated, directly, by ozone (Shallenberger, 2011).

Clinical studies reported the use of prolozone therapy to treat several complications in equine including degenerative joint diseases, sports injuries, and osteoarthritis (do Prado Vendruscolo et al., 2018). In our practice we have performed a total of 2016 treatments as of may 2020. The chart below shows all the anatomical sites where we have used it.

Anatomical Part injected with Ozone

• Back injections
• Cervical Injections
• Medial femoral Condyle Cyst
• ​Mesotherapy treatment with ozone
  
• ​Ozone Ultrasound Guided to masses
  
• ​Cannon bone injection
  
• ​Coffin Joint
  
• ​Fetlock joint
  
• ​Hock joints
  
• ​Pastern Joint
  
• ​Stifle Joits
  
• ​Superficial Digital Flexor Tendon
  
• ​Sacro Iliac Joint
  
• ​Shoulder Joint
  
• ​Suspensory Origin Injection LH prolozone
  
• ​Suspensory Origin Injection RH prolozone
  
• ​Uterine infusion

Equine Hyperbaric Oxygen Therapy

Introduction

The process in which we deliver a high amount of oxygen in a metallic chamber to treat diseased or affected part is called hyperbaric oxygen therapy (HBOT). In the 1960s it was revealed that by placing pigs in a chamber with high pressure of oxygen, their life can be maintained without red blood cells in their vascular system. In the 1990s the animal’s hyperbaric chambers were designed to treat them with hyperbaric oxygen (Weed, Bill, & Gampper, 2003).

For more than 20 years HBOT was only applied to humans for the treatment of injuries and inflammation. In a human, it was started with the treatment of scuba divers with decompression sickness. However, little research is being done for the clinical use of HBOT in animals. Currently, the extrapolation from early animals and humans is being applied to horses to check the physiological effects of HBOT because it seems similar in domestic mammals. In the USA many veterinarians have adopted HBOT for the treatment of infertility and chronic wounds in horses (Geiser, 2016).

The main purpose of HBOT is to increase tissue oxygen concentration to reduce or prevent tissue hypoxia. In many diseases and tissue damage, the oxygen concentration is decreased in the affected area. For this, arterial and capillary oxygen concentration is increased to enrich tissues with oxygen as the diffusion rate between pulmonary capillary blood and alveolus is increased by 4-fold for every 20-fold increased in oxygen pressure. This diffusion rate is increased by two pulmonary effects; high concentration of oxygen in inspired air and decreased alveolar volume by high oxygen concentration in the alveolus (Shah, 2010).

How It Works

During HBOT the metallic chamber is pressurized up to two or three times than normal atmospheric pressure and air is replaced with pure oxygen. The patient is kept placed in this chamber until the unit is depressurized. In horses, two to thirty treatment sessions can be done depending upon the severity of the disease. According to veterinarians’ recommendation, the pressure is increased from 1 to 2.5 ATA (atmosphere absolute) In the first 15 – 20 minutes for normal equine. The next session is of high pressure in the chamber for 45 minutes. After that operator brings back unit to 1 atm. Physicians and veterinarians explain that high levels of oxygen pressure force the oxygen to dissolve in the bloodstream and increase availability to injured tissues. This way the large wounds where oxygen supply has been compromised heal early. HBOT also induce free radical formation in inflammatory cells that kill microorganisms and increase antibodies. The total blood oxygen is increased to 15 times as compared to normal oxygen concentration. This significant increase in oxygen concentration also compensates to low blood flow for tissue oxygenation (Slovis, 2008).

Clinical Applications

HBOT is mainly used as adjunctive therapy but also considered as primary therapy because it provides therapeutic and physiological benefits. HBOT may provide positive outcomes when standard therapeutic measures do not work. Infected cells show a high survival rate in an oxygen-rich environment. The general beneficial effects of HBOT include an increase in tissue oxygen concentration, antioxidant production, fibroblast and collagen production, increased stem cell circulation. HBOT also decreases cerebral edema, cerebral blood flow, selective autoimmune response, and neutrophil-endothelial adherence. Furthermore, it also supports neutrophilic microbial killing, antibodies effectiveness, and aerobic metabolism. HBOT also has a rehabilitation effect on patients without any specificity i.e. it supports tissue regeneration and salvage, the release of stem cells from bone marrow, decrease lipid peroxidation, growth factor stimulation, and vascular neogenesis.

Severe wounds are treated with a combination of laser and hyperbaric oxygen therapy. The combinatorial therapy showed impressive results after 15 days of first treatment. The wound started healing to a great extent. This therapy proves to be useful to treat many complications including the joint diseases, severe chronic infections, laminitis, and lung bleeders etc. (Orsini, 2017)

HBOT has very minute side effects and considered a safe treatment. Patients’ suitability to experience high pressurized environment should be examined, proper screening of patient should be done, and assessments should be performed based on current therapies, diagnosis, pretreatments examination, and diagnosis. Furthermore, the total treatment of equine should be of 90 minutes including pressurizing and depressurizing intervals, the maximum pressure should not be more than 3 ATA and frequency of treatment varies from patient to patient and can reach from 3 to 50 treatments (Heyboer, Sharma, Santiago, & McCulloch, 2017).

Equine Stem Cells Therapy

Equine stem cells are known as adult multipotent stem cells that have the ability to become specialized cells. They are able to regenerate tissues e.g. bone, cartilage, tendon, and muscle. In horses, stem cell therapy is of two types;

• Autologous (patient-derived) Stem Cell Therapy – Isolation of stem cells from your own horses
• Allogeneic (donor-derived) Stem Cell Therapy – Isolation of stem cells from another horse

Stem cell therapy in horses typically involves the use of mesenchymal stem cells (MSCs) that would later form tendons, ligaments, muscles, bones, and fat. These MSCs are mainly isolated/collected from bone marrow, fat or the placenta of the horses. Under specialized conditions, stem cells can also be cultured and replicated in the laboratory (Wei et al., 2013).

Equine Platelets Rich Plasma (PRP) Therapy

Recent research suggests that PRP promotes the healing process by stimulating angiogenesis (new blood vessel formation), improving matrix synthesis, increasing cell migration to damage tissue, proliferation, and differentiation. Several equine clinical studies provide evidence of the significance of PRP therapy that has played a key role in the treatment of tendon and ligament lesions. PRP therapy improves the elasticity and strength of tendons and ligaments while reduces the reinjury rates (Bazzano et al., 2013).

Clinical Applications of PRP Therapy

PRP therapy can be used to treat several equine conditions involving an area where platelets could be injected. These conditions include tendon and ligament injuries, suspensory desmitis, coffin joint collateral ligament injuries, osteoarthritis, bone and joint injuries, and superficial or deep digital flexor tendon (SDFT) lesions, some osteochondral defects and more.

Clinical evidence suggests that the procedure is extremely safe. In a study, the echographic characteristics of the treated tendons were comparable with healthy tendons. The transverse and longitudinal aspects of equine superficial and deep digital flexor tendons (SDFT) showed impressive clinical improvement after PRP therapy as no lesions were seen in the post treated examinations. The subjected horses responded similarly well to the standardized rehabilitation program (Bazzano et al., 2013).

PRP therapy is, undoubtedly, one of the emerging fields of equine regenerative medicine and it holds great potential to change the equine therapeutic dynamics.

IRAP Therapy - ProStride

Introduction

Before we dive into the topic of Prostride. It is important to understand what Autologous Conditioned Serum (ACS) is. The ACS is derived from the horse’s own blood and injected back into the affected part to treat inflammation and several other complications such as degenerative joint diseases.

Interleukin-1 (IL-1) is a cytokine (a substance released by the cells to affect other cells) that is a key mediator of joint diseases. It also plays a significant role in equine musculoskeletal diseases e.g. osteoarthritis. Inhibitors of this cytokine (IL-1) holds a great therapeutic potential against osteoarthritis. IRAP® (Interleukin-1 Receptor Antagonist Protein) is a novel therapy, originally developed in Europe, to treat degenerative joint diseases in horses such as synovitis and osteoarthritis. IRAP prevents the binding of IL-1 on its receptors on tissues with the joint, thereby, blocking the function of IL-1 (Hraha, Doremus, Mcilwraith, & Frisbie, 2011).

Research shows that horses treated with IRAP therapy show a reduction in lameness, improved joint lining, and cartilage preservation (Clarke, Reardon, & Russell, 2015).
Recent clinical evidence suggests that the use of autologous protein serum (APS) was very beneficial. It had significantly improved the values of lameness grade, vertical peak force, and range of joint motion by 14 days when compared to control values. There were no adverse effects observed. The concentration of the interleukin-1 receptor antagonist (IL-1ra) was 5.8 times more in APS than in blood (Bertone et al., 2014).

IRAP therapy is different from other therapies in a way that helps to treat the cause of the joint disease. It aims at restoring the joint lining and function of the cartilage. It is also a long-term treatment for diseases like osteoarthritis, which usually treated with drugs or other therapies that have a short-term effect on the diseased condition.

IRAP therapy is best for those cases that show mild to moderate radiographic signs of the degenerative joint diseases and the lameness is contained to a specific joint/s. It is not recommended to use IRAP therapy to treat tendon sheaths/bursae, in joints with fractures, bone fragments, ligamentous or meniscal injuries before arthroscopic treatment. IRAP is also useful after the removal of chip fragments, arthroscopically. In such cases, proteins and anti-inflammatory cytokines reduce inflammation of the joints and promote regeneration of the damaged cartilage.

ProStride® is a combination of platelet rich plasma (PRP) and interleukin-1 receptor antagonist protein. For simplicity sake, some people have called this product IRAP due it similarity to IRAP®. However it is not exactly the same. In Prostride®, there is a combination of Platelets with autologous conditioned serum creating a dual antinflamatory and regenerative effect.
It is an exclusive dual device system, the output of which produces a concentrated solution of growth factors, cells, platelets, and anti-inflammatory proteins such as IL-1receptor antagonist and other proteins (House & Morton, 2008).

Traditional therapies to treat degenerative joint diseases include the use of nonsteroidal anti-inflammatory drugs (NSAIDs) e.g. Banamine® and Bute. These therapies are being replaced by Regenerative Therapies. PRP therapy and the use of ACS (IRAP) has shown significant clinical success against the treatment of affected tissues and other joint diseases e.g. synovitis (Frisbie, Ghivizzani, Robbins, Evans, & McIlwraith, 2002).

Clinical Applications of ProStride

This therapy can be used for the treatment of osteoarthritis, tendon and ligament injuries, tenosynovitis and some early osteochondritis lesions.
Osteochondritis dissecans (OCD) is a skeletal maturation complication that affects the joint cartilage and the subchondral bone (the bone beneath the surface of the cartilage). OCD can be caused by multiple factors, these include nutritional factors, genetic factors and new evidence suggest that traumatic factors play a bigger part than we thought.

There are three main courses of actions when there are OCD lesions in your horse. The first course of action could be to do nothing and monitor for the lesion to either resolve itself, get worst or to not change. The second course of action would be to performed surgery and clean the affected area. The third and newest technique would be to inject the affected joint with regenerative products and allow it to heal. The advantage of doing regenerative therapies first, is that if the lesion does not disappear, then surgery can always be performed at a later time if needed.

The stifle joint is among the primary joints affected by OCD. It is possible to diagnose stifle OCD in almost every breed. We usually find this disease in young horses because it develops while the bones are forming. However, sometimes it does not bother them until they are older and competing, and other times never causes lameness. It depends in several factors such as the location within the joint and the size. Because we do not know with complete certainty whether the OCD will cause lameness or not, it becomes a conundrum for the team in charge of the horse wether to treat or not.

In this case presented in figure 15, the stifle OCD was treated with Prostride, it showed amazing improvement in joint lining after 90 days followed by near to complete recovery after 150 days from the diseased condition. This is quite encouraging for the clinicians and the horse owners. The back up plan for this patient was removal of the fragment via arthroscopic surgery.

Equine Amniotic Membrane Therapy (AMT)

Introduction

An embryo, when first formed, is closely covered by a membrane known as amnion or amniotic membrane. It is an immune-privileged membrane that contains collagen, several growth factors, extracellular matrix, and some other beneficial proteins known to have anti-inflammatory, anti-fibrotic, anti-bacterial, and re-epithelialization properties. Its structure is quite similar to that of skin. It is filled with a fluid, amniotic fluid, that contains nutrients and growth factors that facilitate fetal growth, provides mechanical cushioning, and serves to aid in the expansion of amnion to become a sac (amniotic sac). This sac protects the developing embryo (Baradaran-Rafii, Aghayan, Arjmand, & Javadi, 2007).

It is biologically relevant in equine regenerative therapies as many therapeutic products are derived from amniotic tissue and fluid. Amniotic tissues when applied to wounds show a significant reduction in scarring and prevents fibrosis during the healing process. Due to their intrinsic biological properties, products derived from amniotic tissues show better results i.e. faster re-epithelialization and reduced healing time as compared to synthetic or biosynthetic products. The biological composition of amniotic tissue may help in the recruitment of patient’s own stem cells to the wound site that greatly enhances the regenerative ability of the body (Dua, Gomes, King, & Maharajan, 2004).

The amniotic membrane, over the last several years, has been used as a dressing to treat equine wounds. This membrane is collected as soon as a foal is born. It seems a bit unusual to employ amnion, yet it is being used in human and equine medicine to treat several diseases including a variety of ulcers, eye complications, burns, and pressure sores. Amniotic membrane, when employed, provides a protective barrier to wounds, prevents loss of protein and fluids, and reduces pain and inflammation at the wound site (Plummer, 2009).

A variety of biological products have been designed using amniotic membrane and fluid to treat several equine complications including soft tissue injuries, laminitis, dermal injuries, topical lacerations, and joint injuries. These products include surgical and resorbable membrane allografts as well as amniotic membrane suspension products. These products can be manufactured and stored at a suitable temperature for later use. Amniotic membrane and fluid is collected from healthy donors during live births without harm to the mare of foal..

Shock Wave Therapy and its Benefits in Horses

Shock Wave Therapy

Shockwave therapy is a comparatively modern non-invasive standard therapy that reduces the risks of surgery and post-surgical discomfort. In the 1980s, this method was first utilized in lithotripsy, a procedure in which strong and energized shockwaves were applied to actively destroy kidney stones in the urinary system.

Massively enhanced bone mineral density within the treated region was noted as an adverse reaction of such lithotripsy therapy, thus resulting in its use in orthopedic surgery, where it has been proven that Extracorporeal shock wave therapies increases osteogenic activation to aid fracture repair. Thus, in the past 2 decades, this one also has been adopted as a treatment for musculoskeletal injuries and improvement of bone density. As a result, these shock waves are applied for the treatment of a variety of orthopedic problems in people, including Achilles tendinopathy, shoulder tendinopathy, elbow tendinopathy, patellar tendinopathy, and plantar fasciitis. In the last few years, shockwave therapies were also used to treat a wide variety of diseases, such as osteochondritis, femoral head necrosis, calcified shoulder tendonitis, and patellar diseases [1-3].

Mechanism

(warning: scientific verbiage can cause sleepiness due to misunderstanding of words).

Shock waves are the highest sound power waves created by a high-voltage blast and absorption beneath the water. Shock waves are applied in lithotripsy to remove nephrolithiasis, whereas in orthopedics they are designed to stimulate neovascularisation (new vessel formation) at the musculotendinous interface and the secretion of chemokines (proteins that stimulate movement and attraction of white blood cells) like vascular endothelial growth factor (VEGFs), endothelial nitric oxide synthase (eNOS), and proliferating cell antinuclear antigen (PCNA). As an outcome, the blood circulation improves cell division, and eventually tissue formation of bones and tendons for the healing process [2, 4].

Shockwaves are defined by a significant and sudden change in pressure, a large amplitude as well as a lack of periodicity. The pressurized air-created kinetic energy of the shotgun blast is passed to the transmitters at the applicator's base and further out into the cells. Energetic waves readily pass forward through the epidermis and into deeper tissues, in which they may accelerate regeneration. This sort of treatment is proven to be safe, non-invasive, minimum cost, and free of the risks of complex surgical procedures and their side effects [5].

Extracellular shockwave treatment seems to be a potential therapy for musculoskeletal diseases in both humans and animals because it is non invasive and avoids several of the complications and expenses of complex surgical treatments. So, a lot of people and animals have used Extracellular shockwave treatment for bone injuries as well as other ligamentous problems in the muscular system [6].

Shock wave therapy for horses

Shockwave treatments help the horse's backbone and several lower limb problems, including joints, tendons and ligaments ailments. It is approved by most horse health insurances and shows excellent outcomes. These waves are an important addition to the armory of therapies available for a diverse variety of injuries. Our capacity to identify diseases has substantially increased in recent years such as ultrasound, digital x-rays, and MRI (magnetic resonance imaging) techniques, while the therapeutic options have remained relatively unchanged over the years until recently. Shockwave technology provides us with an excellent rehabilitation alternative that is less intrusive and has verified effectiveness. In addition, it works excellen with other regenerative therapies such as platelet rich plasma (PRP) and other regenerative therapies.

Extracorporeal shock wave therapy is relatively a recent therapeutic modality for horses with musculoskeletal issues, soft-tissue problems, and bone injuries. Extracorporeal shock wave therapy is a non-invasive technique that promotes healing so that horses can return to full health and functional activity without a less chance of resurgence of illness, if use frequently and in combination with frequent examinations and diagnostics. It repairs tissues rather than just masking pain and swelling. Although, it has also being consider to provide analgesia.

Does it work on wounds? Because of the insufficiency of soft tissue and the stiffness of the surrounding skin, the initial sealing of wounds in the proximal part of the limbs in horses is sometimes difficult [7]. As a result, these wounds are usually healed by secondary intention. However, second intention repair might be exacerbated by the production of excessive granulation tissue, which slows the healing process and leads to a poor visual appearance. Wounds in the distal part of the limbs retract more, and epithelialization occurs more slowly and stops shrinking faster than wounds in the trunk. Several medications and technologies have been proposed to speed up the healing of wounds in the proximal part of the limbs, yet very few controlled trials have been conducted to prove their effectiveness [8].

In this case, extracorporeal shock wave therapy appears to be a recent treatment option that has been found to reduce the time it takes for soft tissue injuries to heal in a wide range of animals. Although the exact mechanism of the faster healing process accelerated by extracorporeal shockwave therapy is not known, it has been observed the increased expression of growth factors such as VEGF, TGF-1, and IGF-1 in the treated tissue. The increased production of these growth factors consequently increases neovascularization, resulting in quicker wound healing [8, 9].

Suspensory desmitis has been successfully stimulated to heal and reduce lameness, according to studies. According to the first clinical findings from two different institutions, seven out of eight and five out of six horses recovered after therapy. Ideally we would like studies with hundreds of horses. By comparing treated limbs to untreated controls in two controlled studies employing a collagenase-induced desmitis model, researchers discovered a reduction in lesion size in treated limbs. Histopathological evaluation revealed that there were more new collagen fibrils and more proteoglycan deposition. Clinical studies have supported such observations, and a retrospective analysis of horses with naturally occurring suspensory desmitis found that extracorporeal shock wave therapy along with regulated exercise was superior to previously described therapies for fore and rear limb proximal suspensory desmitis [10, 11].

How much does shockwave affect pain? A steady voltage, varying current pulse generator was employed in one horse study to measure the horse's nociceptive threshold when the subject firstly responded, and the results showed a minor cutaneous analgesic effect that lasted for three days after extracorporeal shock wave therapy [12].

In summary
Extracorporeal shock wave therapy, which is regarded as a physical therapy/rehabilitation approach, is a useful tool for managing a range of horse conditions/injuries, including:

• Tendinopathy of athletic horses
• Desmitis
• Osteoarthritis including bone spavin
• Stress fractures of the outer section of the cannon bone and partial fractures of the sesamoid bones
• Navicular disease (podotrochleitis)
• Deep muscular pain.
• Back pain due to kissin spine

Alpha-2-macroglobulin

What is Alpha-2-Macroglobulin (A2M)

Alpha-2-Macroglobulin is a family of proteins that are multifaceted and highly conserved among metazoans (animals of metazoan division with body fully developed into tissues and organs). To make it simple for the readr, these are invertebrate animals that evolved from a single cell. The A2M proteins are also present in archaea and bacteria. Although alpha-2-macroglobulin family proteins have many functions in various biological processes, one of the most conserved activities is protease inhibition, which especially helps the body's immune system by neutralizing pathogenic proteases (enzymes involved in breakdown of proteins into simpler amino acids) [1]. In other words, it prevents (inhibit) important proteins from bring broken down or destroyed.

In mammals, the primary site of alpha-2-macroglobulin synthesis is the liver, and it is distributed in the blood, probably to allow better access to all regions of the body, where it majorly contributes to the the innate immune system. Apart from protease regulation, alpha-2-macroglobulin is also involved in the regulation of several other effector molecules as well as the modulation of the host's physiology, thus exert numerous effects on health and in disease progression [2]. Therefore, A2M has an overall important role in the immune system and regular physiology of the animal. 

Mechanism of action (warning: heavy scientific language below)

Alpha-2-macroglobulin gene encodes a protease inhibitor (to prevent the breakn down of proteins) and cytokine (signalling protein) transporter protein. For inhibtion of a wide range of proteases, including trypsis, thrombin, and collagenase, alpha-2-macroglobulin uses a “bait-and-trap” action of amino acids. Alpha-2-macroglobulin acts by developing a tetrameric (structure with four subunits or amino acids) cage enclosing active proteolytic enzymes, ultimately inhibiting the physical interaction between proteolytic enzymes and substrate molecules. See figure one for explanation. This phenomenon can be termed as protease 'snap-trap' or 'venus-flytrap'. Consequently, proteases 'imprisoned' by alpha-2-macroglobulin become unable to cleave larger substrate molecules for example, collagen. While short degradation of short peptide like sneaking into the alpha-2-macroglobulin cage is not affected. The existence of a 'bait region,' which is a string of amino acids and functions as an extraordinarily good substrate molecule for endopeptidases (enzymes that cleave the peptide bond of nonterminal amino acids) of all catalytic forms, helps alpha-2-macroglobulin to preferentially catch exclusively active proteolytic enzymes.  Alpha-2-macroglobulin becomes 'activated' after cleavage of the bait area by a proteolytic enzyme and encounters a structural transformation, therefore 'capturing' active proteolytic enzymes within its tetrameric cage. Alpha-2-macroglobulin after activation exhibits a reactive thioester (molecule with sulfur-acyl group bond) that binds with smaller primary amines in the protease to produce covalent alpha-2-macroglobulin/protease complexes in addition to sterically trapping proteolytic enzymes [3-5].

It can also block the production of cytokines, causing inflammatory processes to be disrupted. Alpha-2-macroglobulin depletion is caused by mutations in its gene. And for its potential to boost the elimination and breakdown of A-beta, the primary component of beta-amyloid plaques, the alpha-2-macroglobulin gene has been linked to Alzheimer's disease (neurological disease) [6].

Benefits of alpha-2-macroglobulin

To this date there is no therapeutic approach available that can cure arthritis at its root. Sigifincat progress has been done towards the slow down, halt, and even reverse joint degeneration after some long-term treatments.

Being a broad-spectrum proteinase inhibitor alpha-2-macroglobulin is commonly found in blood and synovial fluid (fluid present between joints). Its unique molecular structure comprises a bait or trap domain and four-arm configurations (as shown in figure 1), allowing it to inhibit practically all types of proteolytic enzymes. Alpha-2-macroglobulin could trap proteolytic enzymes that cause systemic inflammation. As a result, alpha-2-macroglobulin protects the human body against both endogenous and external inflammatory damages [7]. In both knee osteoarthritic patients and healthy people, however, the concentration of alpha-2-macroglobulin in synovial fluid is found to be substantially lower than that in the bloodstream. Upon this premise, a great number of research studies have looked into the therapeutic impact of intra-articular injections of alpha-2-macroglobulin on knee osteoarthritis. Alpha-2-macroglobulin administration in the knee joint has been shown to successfully postpone the articular cartilage degradation [8-10]. Furthermore, animal models have been used to assess the efficacy of alpha-2-macroglobulin, and the results have validated its protective role [11]. For this reason, it has become so widely used in horses with inflamatioon in their joints.

When combined, alpha-2-macroglobulin and platelet-rich plasma (PRP) therapy provide a potent treatment for osteoarthritis. This method aids in the long-term alleviation of pain related to osteoarthritis [12].

PRP in combination with alpha-2-macroglobulin therapy could provide pain management and better function, allowing patients to resume a healthy lifestyle without the ache and stiffening of osteoarthritis. This combined therapy can enable horses to regain their quality of life and even their athletic capabilities.

The injection of alpha-2-macroglobulin is a cutting-edge novel therapy for the treatment of osteoarthritis and other chronic musculoskeletal diseases. Alpha-2-macroglobulin stimulates the healing process after injecting into an osteoarthritic or sore location because it is a plasma protein molecule that naturally exists in the bloodstream.

As per Dr. Jason M. Cuéllar's study report in human patients, alpha-2-macroglobulin has been used in clinics to treat a variety of orthopedic painful conditions, including subacromial bursitis, lateral epicondylitis, and Achilles tendonitis, with satisfying and promising results. It has also been documented that alpha-2-macroglobulin has therapeutic potential for pain related to degenerative disc disease and enthesopathy (collection of problemtic constions related to rendons and ligaments), a degenerative condition related to knee osteoarthritis. Alpha-2-macroglobulin treatment involves concentrating alpha-2-macroglobulin proteins from donor serum and injecting those into the afflicted body area. To deal with a low concentration of alpha-2-macroglobulin in the synovial fluid, an alpha-2-macroglobulin-rich formulation can be injected into the knee joint of laboratory models, as described in prior studies. Alpha-2-macroglobulin concentrating is now a reality thanks to advanced technologies. A newly designed technique offers a novel approach for concentrating alpha-2-macroglobulin from serum. This concentration system's tangential flow filter technique can concentrate larger proteins such as alpha-2-macroglobulin while filtering out smaller ones. In less than 1 hour, this technique makes alpha-2-macroglobulin-rich concentrations for injection [13].

Despite the fact that a very advanced technique of alpha-2-macroglobulin concentration has already been developed, scientists are still trying to establish a more cost-effective and easy technique. Over 100 tailored variants of alpha-2-macroglobulin have been developed based on its molecular characteristics, 2 of these have the strongest inhibitory action, including CYT-98 and CYT-108 variants [9].

According to the data, the synthesized targeted alpha-2-macroglobulin variants functioned considerably better than the wild-type alpha-2-macroglobulin in protecting joint cartilage against inflammatory destruction. More clinical studies should be conducted to confirm its safety profile in the human body, as well as the large-scale production.

In conclusion, alpha-2-macroglobulin significantly plays role in;

• To prevent cartilage degeneration by halting cartilage breakdown.
• Prevention of osteoarthritis from getting worse.
• Possible joint healing and restoration.
• Reduction of the osteoarthritis associated severe pain.
• Relieving pain while improving joint function.
• Promoting tissue proliferation.

References

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