Known data on applied regenerative medicine in tendon healing

Tendons and ligaments are important structures in the musculoskeletal system. Ligaments connect various bones and provide stability in complex movements of joints in the knee. Tendon is made of dense connective tissue and transmits the force of contraction from muscle to bone. They are injured due to direct trauma in sports or roadside accidents. Tendon healing after repair is often poor due to the formation of fibro vascular scar tissues with low mechanical property. Regenerative techniques such as PRP (platelet-rich plasma), stem cells, scaffolds, gene therapy, cell sheets, and scaffolds help augment repair and regenerate tissue in this context. Therefore, it is of interest to document known data (repair process, tissue regeneration, mechanical strength, and clinical outcome) on applied regenerative medicine in tendon healing.


Background:
Tendon and ligament injuries are quite prevalent in the world. 33 million musculoskeletal impairments are recorded every year where about 50% are linked to tendons and ligaments in USA The current management plan for flexor tendon injuries including the post-operative plan to prevent re-rupture and hypertrophy of tendon is known [20]. However, a meta-analysis found rate of reoperation of 6%, re-rupture of 4%, and adhesion formation of 4% [21]. Achilles tendinopathy accounts for 40 to 50 % of sports injuries in young athletes [22]. Histo pathological studies have proved extensive degenerative changes in ruptured TA [23]. A failure rate of 5%-95% is observed for chronic tears in rotator cuff of shoulder joints [24]. The formation of fibro vascular scar tissue in place of a tough fibro collagenous band [25] due to the presence of antiadhesive protein lubricin in synovial fluid [26] is seen in such cases. Therefore, it is of interest to document known data (repair process, tissue regeneration, mechanical strength, and clinical outcome) on applied regenerative medicine in tendon healing.

Methodology:
The methodology for data collection is illustrated in Figure 1.

Discussion: Methods for tendon repair and regeneration:
Current treatments for tendon repair and augmentation include biological grafts (e.g. auto grafts, allo grafts, and xeno grafts), prosthesis and tissue engineering. The biological grafts have several shortcomings as they induce donor site morbidity (auto graft) and ©Biomedical Informatics (2021) tissue rejection (allograft). However, permanent prosthesis lack material durability causing mechanical malfunctions. Tendon tissue engineering (TTE) represents a most promising approach due to interdisciplinary engineering strategies. It aims to promote full tendon regeneration, rather than physically replacing tendons with partially functionalized foreign substitutes. TTE typically involves scaffolds, stem cells, gels, culture sheets, and gene therapy. TTE scaffolds can enhance tendogenesis by promoting cell proliferation, increasing matrix production, and organizing the matrix into functional tendon tissues. Moreover, tendogenesis can be facilitated through many strategies such as cellular hybridization, surface modification, growth factor cure, mechanical stimulation, and contact guidance.

Figure 2: Stages in tendon healing is shown
Growth factors: Tendon injuries stimulates the increased expression of growth factors particularly in the early phases of healing. The growth factors that have shown a significant impact in tendon healing are bFGF, BMP-12, -13, -14, CTGF (connective tissue growth factor), IGF-1, PDGF, TGFβ, and VEGF. The role of these growth factors in tendon repair is extensively investigated [27-36]. The role of PRP (platelet-rich plasma derivative) has been analyzed in the field of orthopedics over a decade in human (Table 1). PRP is the plasma section of autologous blood containing a large concentration of platelets and growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), fibroblastic growth factor (FGF), and hepatocyte growth factor (HGF) [37]. Most of these factors promote neo-vascularization, tenocyte proliferation, and increase extracellular matrix production. PRP is prepared from autologous blood and it is inherently safe. PRP are present in physiological proportions with a natural balance of proliferative and inhibitory agents [38]. PRP preparation is made simple using advanced preparation devices. These technological advances have allowed PRP treatments to move from operating rooms to outpatient offices produced easily and safely in 15-30 minutes [39-45] ( Table 2).

Scaffolds:
The histological changes that are typical of the healed tendon are poor alterations in fiber structure, arrangement, vascularity, cellular morphology, and cellular proliferation. Scaffolds are placed into the defect zone to provide mechanical support and guide endogenous cells to improve matrix production and organization. give good results in animal models. The findings of these studies are compelling and indicate the need for a long-term evaluation to verify the overall effectiveness of this augmentation method (Table 3).

Tendon gene therapy:
Gene therapy is the utilization of therapeutic nucleic acids into patient's cells to treat a disease condition. Tashjian et al. identified an SNP within the estrogen-related receptor beta (ESRRB) gene that appears to promote increased susceptibility to re-tears after a rotator cuff repair . Current tissue engineering strategies using synthetic biomaterial scaffolds have yet to yield tendon substitutes. The appeal of these engineered scaffolds is that they can potentially be impregnated with growth factors or genes for targeted and timed-release at the site of implantation to improve healing. We reviewed 9 studies (Table 4) for the effect of various genes (rAAV-Gdf5, BMP-12, BMP-14 and PDGF) on tendon healing, strength, and movement [60][61][62][63][64][65][66][67][68][69]. This data is promising for further consideration.

Stem cells:
Pluripotent stem cells carry great potential for cell therapy and tissue engineering. The use of embryonic stem cells (ESCs), adult mesenchymal stem cells (MSCs) tendon derived stem cells (TDSs), and Human skeletal muscle progenitor (SMP) cell to regenerate functional tendons and ligaments [70][71][72][73][74][75][76][77][78][79] (Table 5) is of interest. Various sources of MSCs have been investigated for their impacts on tendon repair. Embryonic stem cells (ESCs) have unlimited proliferation capacity and it can be induced into all types of somatic cells for tissue repair. However, there is a risk of teratoma formation. There are two promising cell types, namely bone marrow mesenchymal stem cells (BM-MSCs) and adipose-derived mesenchymal stem cells (AD-MSCs). They are well characterized and simple for in vitro proliferation. Interestingly, most of the preclinical animal studies concluded that MSC delivery can lead to increased cell proliferation, but these cells often differentiated towards osteoblasts or adipocytes within the tendon area, suggesting their inherent preference to commit to the original lineage of the tissue from which they were isolated [80]. The isolation of the native to the tendon-tenocytes, tendon stem/progenitor cells, or tendon-derived fibroblasts is relevant to the context [81]. MSCs have self-renewal and multilineage differentiation potential. BMSCs have shown immense collagen production after seeding on polylactide/glycolide (PLGA) suture material. Lee et al. [77] used Allogeneic adipose-derived mesenchymal stem cells in lateral epicondylosis and found tendon defect significantly reduced in 6 weeks. Ilic et al. studied mesenchymal stromal cells (MSCs) from the human placenta. They were injected directly into the site of tendon damage using ultrasound guidance in the treatment of chronic refractory tendinopathy and observed that there is significant improvement in tendon repair. Hernigou et al. [79] showed the role of crest bone marrow-derived mesenchymal stem cells (MSCs) in rotator cuff injury to prevent further damage.

Gel and cell sheets:
Tendon repair and minor defects can be augmented with hydrogels with stem cells or direct cell sheets ( Table 6). The tendon hydrogel promotes host cell infiltration, supporting its biocompatible properties and sustained the viability and proliferation of donor, adipose-derived stem cells (ASCs). The tendon hydrogel's thermoproperty under physiologic temperature enhances its applicability in vivo. The gel polymerized and formed the shape of the defect at 37 degree Celsius. Hydrogel is a promising biomaterial for guided tissue regeneration. Degen et al. [82] showed rotator cuff repair augmentation with purified human MSCs with hydrogels in rat models. It was observed that there is improved early histologic appearance and biomechanical strength of the tendon at 2 weeks as described elsewhere [83-86]. Cell-cultured sheets derived from adipose stem cells, ACL, rotator cuff, and tendon stem cells were also used in this context despite increased cost [87][88][89][90][91].

Amniotic membrane:
The epithelial and mesenchymal cells of amnion contain various regulatory mediators like Epidermal growth factor, Keratinocyte growth factor, a hepatocyte growth factor that results in the promotion of cellular proliferation, differentiation, epithelialization, inhibition of fibrosis, immune rejection, inflammation, and bacterial invasion ( Table 7) [92]. The presence of platelet-derived growth factor (PDGF) and vascular endothelial-derived growth factor (VEGF) is suggestive of a pro-angiogenic role [93]. It is known that amniotic epithelial and mesenchymal cells lack HLA class A, B, DR, and co-stimulatory molecules CD-40, CD-80, and CD-86 making it non-immunogenic [94]. The effects of human amniotic fluid on peritendinous adhesion formation and tendon healing after flexor tendon surgery in rabbits are shown [95]. Amniotic membrane in flexor tendon repair has reduced adhesion [96]. Properties of the amniotic membrane for potential use in tissue engineering are available [97]. Flexor tendon repair using allograft amniotic membrane is also shown [98, 99].

Conclusion:
Known data (repair process, tissue regeneration, mechanical strength, and clinical outcome) on applied regenerative medicine in tendon healing is documented in this review. Information on the use of applied regenerative technologies such as the use of growth factors, scaffolds, gene therapy, stem cells, gel and cell sheets and amniotic membrane in tendon healing is gleaned from known literature to enrich our knowledge in this context. Caveats and limitations on known data including clinical trials, evidence based research information and FDA reveiws were found to be useful for further consideration [100-104].

Conflict of Interest:
There is no conflict of interest in this article.

Ethical approval:
The Ethical committee of MMMCH at Kumarhatti Solan approved the review material. [  (4): 514-527 (2021) License statement: This is an Open Access article which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. This is distributed under the terms of the Creative Commons Attribution License In vitro PRP is a source of growth factors such involved with tendon-bone healing. PRP had an anabolic effect on the human rotator cuff tenocytes of the same individual in vitro by means of cell proliferation and absolute, but not relative collagen I synthesis. These results encourage further studies on clinical outcomes with more comparable standards in terms of preparation and application methods.

References
Stephan Pauly et al. 2012

PRP Allogenic PRP gel In vitro
The results of this study suggest that the local application of PRP could enhance the tissue-healing process both directly through action on localized cells and indirectly through the recruitment of reparative cells through the blood flow. Further investigations will be needed to confirm the mechanisms of PRP in tissue-healing processes with the development of this experimental model.

Yohei
Kobayashi et al. 2020 6. PRP PRP combined with recombinant human type 1 collagen In vitro STR/PRP is a safe treatment that effectively induces clinically significant improvements in elbow symptoms and general well being as well as objective measures of strength and imaging of the common extensor tendon within 6 months of treatment of elbow tendinopathy recalcitrant to standard treatments.
Uri Farkash et al. 2019 Rabbit; patellar tendon; full-thickness surgical defect; PRP with the gel form were placed in the defect; analysis at 1, 2, 3 and 4 wks.
Stronger and more extensive expression of TGF-b1 was showed at 1 and 2 wks by immunohistochemistry.

Growth factors
Basic fibroblast growth factors Rat; Achilles tendon; surgical defect; analysis at 12 weeks Biomechanical properties were not significantly improved.
Kraus et al.

Growth factors
Bone morphogenetic Protein 12 (BMP 12) Dog; flexor digitorum profundus tendon; surgical transection; 5 mm depth, 2.5 mm width; scaffold with adipose derived stromal cells and BMP 12 were placed in the transection; analysis at 28 days.
Tensile properties showed no significantly difference; Proteomics analysis showed amplification of Inflammation, stress response and matrix degradation Gelberman et al.

Growth factors
Bone morphogenetic Protein 2 (BMP 2) Rabit: First, recombinant human bone morphogenetic protein-2 (rhBMP-2) was injected into the flexor digitorum communis tendon in the rabbit hind limb to induce ectopic ossicle formation. In a second step, the resultant tendon/ossicle complex was then surgically transferred onto the surface of the rabbit tibia to generate a stable tendon-bone junction.
Enthesis like tissue had been successfully formed at 4 weeks and this tissue was shown functionally competent for mechanical repairing Hashimoto G et al.

Growth factors
Insulin growth factor -1 (IGF-1) Rabit: the effects of recombinant human insulinlike growth factor (rhIGF-I), insulin and fetal calf serum The E max of stimulation of proteoglycan and collagen synthesis by rhIGF-I were two times that of FCS, and Abrahamsson SO et al.
©Biomedical Informatics (2021) (FCS) on the synthesis of proteoglycan, collagen, and non-collagen protein and cell proliferation were investigated in short-term explants cultures of the deep flexor tendon the E max of cell proliferation by FCS was twice that of rhIGF-I. Growth factors thus have the ability to stimulate matrix synthesis and cell proliferation in rabbit flexor tendon. 6.
Growth factors Platelet-derived growth factor (PDGF) Rat: Platelet-derived growth factor isoform B at various dosages (0, 10, 100, or 1000 ng) was delivered into the gap wound in patellar tendons via microsyringe injection on Day 3 or Day 7 after injury. Tendon specimens were harvested on Day 14 for measurement of cell proliferation, pyridinoline content, and mechanical properties.
Supplementation of platelet-derived growth factor isoform B at Day 7 benefits the mechanical properties and maturation of healing tendons.
Chan BP et al.

Growth factors
Transforming growth factor (TGF) Rabit: 30 female rabbits were divided into three groups, after a 3 mm wide and 10 mm long tendon substance was resected from the central portion in the patellar tendon. In Group I, 5-ng TGF-beta1 dissolved in 0.1-ml saline was injected into the resected portion in the patellar tendon.
In Group II, only 0.1-ml saline was injected into the resected portion. In Group III, nothing was injected. All animals were sacrificed at 6 weeks after surgery Rat: the Achilles tendons of 80 Sprague Dawley rats were transected and sutured back together. Ten rats from each group (ACS group, n = 40; control group, n = 40) were euthanized at 1, 2, 4, and 8 weeks postoperatively for biomechanical (n = 7) and histologic (n = 3) testing.
The ACS-treated tendons were thicker, had more type I collagen, and an accelerated recovery of tendon stiffness and histologic maturity of the repair tissue Majewski et al. Defects receiving collagen sponges showed improved healing, with significantly stronger and less stiff tendons than control tendons. No inflammatory reaction due to the collagen sponge was found histologically.

Biomaterials
Ovine forestomach matrix (OFM) scaffold Rat; rotator cuff, surgical transection; OFM scaffolds (5 mm × 10 mm) were overlaid longitudinally on the superficial aspect of the tendon-bone insertion; analysis at 6 days and 12 wks Improved healing quality was shown by histological analysis, no evidence of excessive inflammatory response, no biomechanical advantage of augmentation Street et al.

Biomaterials Porcine small intestinal submucosa (SIS)
Human-2-year followup of 12 patients who underwent arthroscopic repair of massive chronic rotator cuff tears using Restore SIS as an augmentation device.
Postoperative magnetic resonance imaging (MRI) scans showed significant thickening of the cuff tendon with the incorporation of the SIS graft in 11 patients.
Metcalf et al.

Biomaterials
Collagen Repair Patch (single layer porcine skin xenograft) Human-evaluated 10 patients with extensive rotator cuff tear treated with Zimmer Collagen Patch All patients experienced significant pain relief and improvement in abduction power and range of motion. Ultrasound imaging at the final follow up identified intact grafts in eight and disrupted grafts in two patients Badhe et al.

Biomaterials Acellular dermal
Human-11 patients with acute tendon ruptures At 20 months, there were no reruptures or recurrent pain; Lee Dk et ©Biomedical Informatics (2021) matrix (ACM) were followed up for 20 to 31 months with ACM the average return-to-activity time was 11.8 + 0.75 weeks. Significant increase in strength and stiffness of Achilles tendon repair augmented al.

Biomaterials Fresh autograft fascia lata
Rabbit-in supraspinatus injury model fresh autograft fascia lata as an interpositional graft At the fascia-bone junction, chondrocytes started to appear at 2 weeks after surgery, and increased rapidly in number and columnar organization. By 8 weeks, remodelling of direct insertion with fibrocartilage was almost complete Sano et al.

Synthetic Scaffolds
Polyglycolic acid (PGA) sheet Rabbit: polyglycolic acid (PGA) sheet to augment rotator cuff repairs of infraspinatus tendons Histological improvement in fibrocartilage layering but only a slight improvement in tensile strength Yokoya et al.

8.
Biomaterials Synthetic ECM Rabbit-in infraspinatus tendons the 10-mm defect was covered with chitin, a biodegradable polymer, sutured into the bone trough, and attached to the free end of the infraspinatus tendon. The contralateral shoulder was left untreated as a control.
The tendon-to-bone junctions covered with chitin fabric demonstrated greater cell number, better collagen fiber alignment, and greater mechanical strength than the tendon-to-bone junctions left free as control 9. Biomaterials Polylactic acid patches Dog-The superior 2.3 of each infraspinatus tendon was removed from the rotator cuff and then repaired in both shoulders. In one shoulder, a woven poly-Llactide device was placed over the repair. In the other shoulder, the repair was left unaugmented.
The augmented rotator cuff repair resulted in fewer tendon retractions, greater strength, and increased stiffness when compared to the contralateral untreated rotator cuff repairs Derwin et al.

Biomaterials Polymer filamentous carbon composites
Human-implant composed of filamentous uniaxially aligned carbon fibres coated with an absorbable polymer in 48 patients with a rupture of Achilles tendon.
The early strength of this repair was provided by the composite implant and by the rapid ingrowth and attachment of new tissue. All patients demonstrated continuous improvement during the first post-operative year, and a high level of function throughout the second year. Both repair of chronic and acute injury greatly improved Parsons et al. In vivo BMP-14/GDF-5 with AAV Mouse, flexor tendon: recombinant adenoassociated virus (rAAV)-loaded tendon allografts mediate efficient transduction of adjacent soft tissues, with expression peaking at 7 days The rAAV-Gdf5 vector significantly accelerates wound healing in an in vitro fibroblast scratch model and, when loaded onto freeze-dried FDL tendon allografts, improves the meta tarso phalangeal (MTP) joint flexion to a significantly greater extent than the rAAV-lacZ controls do.
Basile P et al.

BMP-14/GDF-5 Adenovirus
Rat Achilles: the histological and biomechanical effects of adenovirusmediated transgene expression of bone morphogenetic protein-14 (BMP-14) on healing in a rat Achilles tendon laceration model ©Biomedical Informatics (2021) vector harboring the basic fibroblast growth factor gene were injected into both ends of the cut tendon. In Group 2, the same amount of adeno-associated viral vector carrying the luciferase gene was injected. In Group 3 (the non-injection control group), the tendons were sutured without any injection. viral vector-basic fibroblast growth factor was significantly greater than that of tendons that had been treated with the sham vector or simple repair both during the early healing period four weeks, and a later period of eight weeks Ex vivo 7.
Hou Y et al.

Scleraxis with Adenovirus
Rat, superspinatus: Thirty animals received MSCs in a fibrin glue carrier, and 30 received Ad-Scx-transduced MSCs. Animals were sacrificed at 2 weeks and 4 weeks and evaluated for the presence of fibrocartilage and collagen fiber organization at the insertion. Biomechanical testing was performed to determine the structural and material properties of the repaired tissue There were no differences between the Scx and MSC groups in terms of histologic appearance at 2 weeks. However, the Scx group had higher ultimate stress-to-failure and stiffness) compared with the MSC group.

BMP-12/GDF-7 Adenovirus
Rat, Achilles: Biopsies of autologous skeletal muscle were transduced with a type-five, firstgeneration adenovirus carrying the human BMP-12 cDNA (Ad.BMP-12) and surgically implanted around experimentally transected Achilles tendons in a rat model. The effect of gene transfer on healing was evaluated by mechanical and histological testing after 1, 2, 4 and 8 weeks Reatment with BMP-12 cDNA-transduced muscle grafts thus produced a promising acceleration and improvement of tendon healing, particularly influencing early tissue regeneration, leading to quicker recovery and improved biomechanical properties of the Achilles tendon.
Majewski M et al.  TDSCs showed better biomechanical properties and higher tendency in Col-I/III gene expression level during wks 1 and 2. Immunofluorescent assay revealed higher expression of Tenascin-C in TDSCs at week 1.
Al-ani et al.

Horse amniotic membrane-derived mesenchymal cells (AMCs)
Horse: the immunomodulatory characteristics of AMCs and of their conditioned medium (AMC-CM) in vitro, and studied the potential therapeutic effect of AMC-CM in thirteen different spontaneous horse tendon and ligament injuries in vivo.
AMCs are capable of inhibiting peripheral blood mononuclear cell (PBMC) proliferation after allogenic stimulation either when cocultured in cell-to-cell contact and Clinical outcomes were favorable and the significantly lower rate (15.38%) of reinjuries observed compared to untreated animals 5.
Human skeletal muscle progenitor (SMP) cell Mouse: The SMP population was quantified, isolated, and assayed in culture for its ability to proliferate and fuse in vitro and in vivo. Cells from all cuff states were able to fuse robustly in culture and engraft when injected into injured mouse muscle SMPs are capable of contributing to muscle hypertrophy and regeneration regardless of tear severit Gretchen A Meyer et al.
The treated TDSCs accelerated and enhanced the quality of tendon repair compared with untreated TDSCs up to week 8, which was better than that in the controls up to week 16 as shown by histology, ultrasound imaging and biomechanical testing.
Lue et al.

Mesenchymal stem cells (MSCs)
Rabbit; they were divided into 6 groups (three treatments with two time points each) evaluated at either 14 or 28 days after surgery: cross section of the Achilles tendon (CSAT); CSAT + Suture; and CSAT + MSC.
Comparison between the two time points within the same group showed a statistically significant decrease in the inflammatory process and an increase in the structural organization of collagen in the CSAT and CSAT + MSC groups MSC transplantation is a good alternative for treatment of Achilles tendon ruptures Vieira MH et al.

8.
Allogeneic adiposederived mesenchymal stem cells Human: lateral epicondylosisllo-ASCs mixed with fibrin glue were injected into the hypoechoic common extensor tendon lesions all evaluated at 6, 12, 26, and 52 Tendon defects also significantly decreased through this period. Allo-ASC therapy was thus safe and effective in improving elbow pain, performance, and structural defects for 52 weeks.
Lee et al.

9.
Mesenchymal stromal cells (MSCs) from Human placenta Human:MSCs were injected directly into the site of tendon damage using ultrasound guidance in the treatment of chronic refractory tendinopathy.
Clinical trials using both allogeneic and autologous cells demonstrated MSCs to be safe.
Ilic N et al.
Human: forty-five patients in the study group received concentrated bone marrow-derived MSCs as an adjunct to single-row rotator cuff repair at the time of arthroscopy. The average number of MSCs returned to the patient was 51,000 ± 25,000.
Forty-five (100 %) of the 45 repairs with MSC augmentation had healed by six months, versus 30 (67 %) of the 45 repairs without MSC treatment by six months. Bone marrow concentrate (BMC) injection also prevented further ruptures Hernigou P et al. ©Biomedical Informatics (2021) 525 BMSCs augmented with either fibrin glue (control group) or fibrin glue with 10 6 human MSCs (experimental group) applied at the repair site.
biomechanical strength of the repair at 2 weeks, although the effects dissipated by 4 weeks with no significant differences between groups. 2.
Fibrin TSPCs Rat: Green fluorescent protein-TDSCs (GFP-TDSCs) were pre-treated with or without CTGF and ascorbic acid for 2 weeks before transplantation. The patellar tendons of rats were injured and divided into three groups: fibrin glue-only group (control group), untreated and treated TDSC group. The rats were followed up until week 16.