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Jennifer Lei, PhD, Lauren B. Priddy, PhD, Jeremy J. Lim, PhD, and Thomas J. Koob, PhD


The placenta plays a critical role in transporting nutrients and protecting the fetus during pregnancy. The developing fetus and amniotic fluid are enclosed in the amniotic sac of the placenta, circumscribed by the amniotic membrane, which functions to help modulate the contents of the intra-amniotic compartment and mediates the interaction between the fetal and maternal tissues. The amniotic membrane is composed of 2 distinct layers that surround the embryo: the amnion that lines the inner surface of the amniotic sac interfacing with the fetus, and the chorion that is the outer layer in contact with maternal tissue. The amnion layer is composed of an epithelial layer, a basement membrane, a compact layer, and a fibroblast layer. Collagens type I and type III are located in the fibroblast layer and provide most of the tissue’s mechanical strength. Collagen type V and type VI create filamentous cross-links between the fibrous network and the basement membrane that contains collagen type IV. Other extracellular matrix (ECM) components, such as fibronectin and laminin, are found throughout the amnion tissue. An intermediate spongy layer separates the amnion and chorion with a jelly-like network rich in collagen III, proteoglycans, and glycoproteins. 


The chorion layer is composed of a reticular layer containing collagens I, III, IV, V, and VI and proteoglycans; a basement membrane layer containing collagen IV, fibronectin, and laminin; and a trophoblast layer. Together, the amniotic membrane is a fibrous tissue containing many ECM components. Some of the many factors present in this bioactive ECM include platelet-derived growth factors (PDGFs), transforming growth factor-b1 (TGF-b1), basic fibroblast growth factor, and granulocyte colony-stimulating factor, as well as interleukin-4 (IL-4), IL-6, IL-8, and IL-10.7 The crucial roles of this metabolically active tissue are to function as a barrier for water and soluble material transport, to synthesize growth factors and cytokines, and to regulate amniotic fluid pH.

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Native ECM materials such as amniotic membrane represent a class of naturally derived biomaterials already used in the clinic for tissue healing applications. Amniotic membrane was used as early as 1910 for skin grafting and has been used successfully for healing of various tissues including skin, cornea, and ligament in clinical settings, as well as cartilage and cardiac repair in preclinical animal models. In addition to being nutrient-rich tissues with the ability to stimulate soft tissue development, amniotic membrane acts as a nonimmunogenic barrier between the mother and fetus during pregnancy; therefore, amniotic allograft tissue can be transplanted without rejection by the host, as observed in clinical applications. Typically, amniotic membrane is harvested upon scheduled cesarean section from donors who undergo rigorous screening for viral and infectious diseases, including human immunodeficiency virus 1 and 2, hepatitis B and C, human T-cell lymphotrophic virus, and syphilis.


After washing and cleaning harvested tissues in buffered solutions, amniotic membrane grafts can be prepared as fresh or preserved allografts. When using fresh amniotic membrane grafts, immediate transplantation is often necessary; therefore, preservation techniques such as cryopreservation or dehydration have been utilized to increase storage time. Although both preservation methods have exhibited diminished maximum loads to failure and elongation to failure in tension compared with fresh tissue, the structural components and integrity of the tissue remain intact with both techniques.



Orthopedic soft tissue healing occurs in similar process to wound healing. The 3 main phases are the inflammatory phase, the proliferation phase, and the remodeling phase. The initial inflammatory phase occurs immediately after the injury and can last up to 1 to 3 days, during which increased blood flow helps increase fibrin clot formation and form a mesh to trap any foreign particles and debris. Mast cells and neutrophils are recruited to the site to help phagocytose damaged tissue or debris and release cytokines, such as tumor necrosis factor-a, IL-1a, IL-1b, TGF-b1, granulocyte colony-stimulating factor, and macrophage colony-stimulating factor. These cytokines help recruit other inflammatory cells, such as monocytes/ macrophages and lymphocytes, that stimulate the proliferative phase of the healing process.


The proliferative phase begins 24 to 48 hours after injury and persists for 2 to 3 weeks. During the proliferation phase, resident tissue cells migrate to the area from surrounding tissues and are activated by factors released by local macrophages. Upon activation, these cells, which include fibroblasts for fibrous tissues, proliferate and secrete disorganized collagen-based tissue. In addition, secretion of factors such as macrophage-derived factors, PDGF, and FGF result in the formation of developing capillaries and angiogenesis of the scar tissue. Resolution of tissue healing ends with the remodeling phase that results in the formation of organized collagen fibers that resembles the original tissue as closely as possible. The initial deposition of collagen fibers during the proliferative phase are relatively weak fibrils; therefore, during remodeling, the fibers organize and align themselves in the direction of local stresses to create a fibrous tissue that may not exactly recapitulate the original tissue structure, but does restore full functional utility.  Although tissue healing is a complex process, therapies that can intervene or support such events can be beneficial to the repair process to produce the best quality tissue in minimal time.


Appropriate interventions, such as modulating the inflammatory response to maximize recruitment of cells or refining the scar tissue formation during the proliferative and remodeling phases, can be used to effectively produce a healed tissue that best resembles the structure and function of the original tissue before injury. 

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Typically, for orthopedic tissue injuries, current repair techniques often only address restoring mechanical function by reattachment or replacement of the tissue. For example, tendon and ligament repair in knee joints use autografts or allografts to physically reconnect the tissues. Although the patient may exhibit decreased pain and return to functional usage at the site of injury, the biological structure of that tissue is not reproduced, which can increase the potential for future reinjury. By using alternative methods to both restore function and regenerate tissue with structure similar to that of its native form, both repair and regeneration of injured orthopedic tissues can potentially be achieved.


Alternative methods of repairing orthopedic tissues involve the application of bioactive factors, such as tissue engineering strategies using growth factors, scaffolds and amnion membrane, that can promote healing and regeneration of the tissue itself. Amnion’s Cytoflo, injected directly into the affected area promotes rapid growth while reducing inflammation. This has been tested and shown to be highly effective in more than 400,000 cases. The human amnion derived tissue (HADT) is a natural ECM biomaterial that contains many growth factors, cytokines, proteases, and regulatory inhibitors that can contribute to the process of soft tissue healing. HADT allografts have shown clinical success in healing poorly vascularized chronic wounds and therefore have promising potential to also promote healing in less vascularized orthopedic tissues like tendon, ligament, and cartilage. Owing to these characteristics, HADT allografts have recently been used in soft orthopedic tissues and spine applications to reduce pain, prevent scar tissue formation, and promote healing.  



The incorporation of HADT tissues can decrease fibrous collagen deposition scar formation in vitro and modify inflammatory responses of tenocytes. Compared with adult wound healing, fetal wound healing has the ability to form highly aligned and organized fibers with minimal scar formation, suggesting that fetal tissues and the fetal environment may be uniquely capable of supporting tissue regeneration. Therefore, one approach to recapitulate fetal healing is to use ECM-based biomaterials that originate from environments with anti-inflammatory and antimicrobial properties, such as amniotic tissue. It was shown that when amniotic membrane tissue was incorporated into tenocyte-laden collagen-glycosaminoglycan scaffolds, cells exhibited increased metabolic activity in both basal and proinflammatory environments (induction with IL-1b) compared with scaffolds without amniotic tissue. In addition, the addition of amniotic membranes also down regulated the gene expression of the proinflammatory molecules tumor necrosis factor-α and matrix metalloproteinase-3 in tenocytes, indicating that this biomaterial could alter the inflammatory response associated with scar formation in tendon healing to better mimic fetal soft tissue healing.


Methods of incorporating hyaluronic acid (HA) have also been explored to reduce scar formation, as HA is known to play a role in chronic wound healing by promoting cell proliferation and motility. As a critical component of several orthopedic tissues including cartilage and synovial fluid, HA contributes both mechanical properties as well as the ability to regulate cellular activity through interaction with growth factors and binding of cell surface receptors, such as CD44. In particular, HA is an ECM component that has been detected and quantified in HADT tissues and may play a role in improved soft tissue healing.  Thus, the use of amniotic membranes that contain HA could potentially be an effective method to help modulate the inflammatory environment to decrease scar formation during tendon and ligament healing. In flexor tendon transection models in chickens, it has been shown that the use of amniotic membranes can improve flexor tendon repair.  Zone II flexor tendon injuries can lead to loss of hand functions due to the formation of fibrous adhesions and restriction of tendon gliding.


One approach to prevent tissue adhesion formation is to use membranous materials, such as amniotic membrane allografts, which can act as a barrier between the healing tendon and the surrounding tissue environment. In white leghorn chickens, digital flexor tendons were incised and repaired with a modified Kessler stitch, and amniotic membrane was sutured to the tendon proximally and distally away from the cut ends, ultimately surrounding the repaired tendon tissue. By week 12, histologic analysis revealed that tendons covered with the amniotic membrane did not exhibit granulation tissue or fibrous adhesions, as was observed with groups without amniotic membrane intervention. In addition, organized, aligned collagen fibers were observed throughout the healed tendon. Using the Tang scale to evaluate adhesion formation, it was determined that amniotic membrane coverage was beneficial in preventing adhesion when compared with repaired tendons without the graft. Collectively, these data demonstrate that the amniotic membrane can be used to assist in the treatment of reconstructed tendons and prevention of adhesions. Owing to the many growth factors contained in the HADT tissue, biomolecules such as EGF, TGF-b, FGF, and PDGF-AA and PDGF-BB may stimulate cell migration and proliferation, as well as metabolic processes such as collagen synthesis to help initiate tendon healing.  In another case study, a HADT allograft patch was used to supplement a ruptured ACL that was reconstructed using a hamstring autograft. 


During the arthroscopic ACL reconstruction procedure, the hamstring autograft was augmented with a HADT allograft patch fixated using the Tape Locking Screw (TLS) technique. The autografts were wrapped in HADT and rolled around 2 posts to form a 4-strand closed loop with TLS strips passed through 2 ends of the tendon loops. Subsequent magnetic resonance imaging scans revealed vascularization in the hamstring graft tissue as early as 3 months post-operative and the patient’s rehabilitation progressed successfully with regards to strength and proprioception at 8 months post-operative. Although the sample size was small for this study, this represents another example of HADT uses in clinical practices for treatment of tendon and ligament injuries. Taken together, recent preclinical and clinical findings suggest that the administration of HADT allografts is a viable option to treat tendon and ligament injuries.

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Amniotic membrane-based allografts are classified as a naturally derived biomaterial and are currently being used in wound and soft tissue repair applications. Amnion’s Cytoflo has been used in more than 400,000 cases with great success. Characterization of this tissue has revealed many growth factors, cytokines, and protease inhibitors contained within HADT tissue that can play a role in wound repair. HADT has been shown to stimulate cellular activity including proliferation, migration, and secretion of soluble paracrine factors in vitro, and has also demonstrated the ability to recruit reparative adult stem cells to the site of HADT implantation in vivo. In addition, these allografts have been shown in randomized clinical trials to be effective in healing various dermal and soft tissue wounds. Because of their regenerative properties, HADT may also be used to repair and regenerate orthopedic tissues. To date, amniotic membrane and HADT have been evaluated for repair of tendon and ligament, attenuation of cartilage and joint space diseases, and prevention of scarring and adhesion formation in spinal fusion procedures.


HADT allografts can be used both as a therapy to decrease pain and reduce fibrous tissue formation, for example in plantar fasciitis treatment, or as a supplement patch to a current procedure being performed, for example in ACL reconstruction. Clinical usage in orthopedic repair is rapidly growing and many ongoing clinical trials using HADT allografts in plantar fasciitis treatments, Achilles tendon repair, lumbar decompression and microdiscectomy and total knee arthroplasty are underway (  Current research and clinical cases using amniotic membrane for repairing orthopedic tissues have shown that HADT allografts can have promising results in repairing injured and diseased tissues due to their ability to deliver a natural ECM biomaterial that contains many active biomolecules. There is great potential for the use of amniotic membrane allografts for regenerative applications in orthopedics; however, much more research will be necessary to specifically define what those applications will be.