Autologous bone marrow derived mesenchymal stem cells are safe for the treatment of Achilles tendinopathy.

Achilles tendinopathy is a disabling condition that affects more than 50% of runners. Pre-clinical studies in a large animal model of naturally-occurring tendinopathy similar to human Achilles tendinopathy has shown benefits of autologous bone marrow-derived mesenchymal stem cell (MSC) implantation. However, MSCs are advanced therapies medicinal products (ATMPs), with strict regulatory requirements. Guided by the regulator we carried out a first in man study to assess the safety and efficacy of autologous MSC injection in human patients with non-insertional Achilles tendinopathy. Ten patients, mean age 47 with mid-portion Achilles tendon pain and swelling for more than 6 months, underwent autologous cultured cell injections (median 12.2 × 106, range 5-19 × 106 cells) into their Achilles tendon. At 24 weeks follow-up, no serious adverse reactions or important medical events were observed. MOXFQ, EQ-5D-5L, and VISA-A scores improved clinically at 12 and 24 weeks. VAS pain improved increasingly at 6, 12 and 24 weeks. MOXFQ Pain and VISA-A Scores improved > 12 points from baseline to 24 weeks in 8 patients. Maximum anteroposterior tendon thickness as measured by greyscale US decreased by mean 0.8 mm at 24 weeks. This phase IIa study demonstrated the safety of autologous MSC injection for non-insertional Achilles tendinopathy and provides proof-of-concept of the technique in patients, all of whom had previously failed conservative treatments for chronic disease and leads the way for a larger randomised controlled trial.

Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell-derived factors.

Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1ß, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells' ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1ß using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1ß stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB.

Histopathological and immunohistochemical evaluation of cellular response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch for tendon repair.

We investigated endogenous tissue response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch intended for tendon repair. A sheep tendon injury model characterised by a natural history of consistent failure of healing was chosen to assess the biological potential of woven and aligned electrospun fibres to induce a reparative response. Patches were implanted into 8 female adult English Mule sheep. Significant infiltration of tendon fibroblasts was observed within the electrospun component of the patch but not within the woven component. The cellular infiltrate into the electrospun fibres was accompanied by an extensive network of new blood vessel formation. Tendon fibroblasts were the most abundant scaffold-populating cell type. CD45+, CD4+ and CD14+ cells were also present, with few foreign body giant cells. There were no local or systemic signs of excessive inflammation with normal hematology and serology for inflammatory markers three months after scaffold implantation. In conclusion, we demonstrate that an endogenous healing response can be safely induced in tendon by means of biophysical cues using a woven and electrospun patch.

Bone marrow mesenchymal stem cells do not enhance intra-synovial tendon healing despite engraftment and homing to niches within the synovium.

BACKGROUND: Intra-synovial tendon injuries display poor healing, which often results in reduced functionality and pain. A lack of effective therapeutic options has led to experimental approaches to augment natural tendon repair with autologous mesenchymal stem cells (MSCs) although the effects of the intra-synovial environment on the distribution, engraftment and functionality of implanted MSCs is not known. This study utilised a novel sheep model which, although in an anatomically different location, more accurately mimics the mechanical and synovial environment of the human rotator cuff, to determine the effects of intra-synovial implantation of MSCs. METHODS: A lesion was made in the lateral border of the lateral branch of the ovine deep digital flexor tendon within the digital sheath and 2 weeks later 5 million autologous bone marrow MSCs were injected under ultrasound guidance into the digital sheath. Tendons were recovered post mortem at 1 day, and 1-2, 4, 12 and 24 weeks after MSC injection. For the 1-day and 1-2-week groups, MSCs labelled with fluorescent-conjugated magnetic iron-oxide nanoparticles (MIONs) were tracked with MRI, histology and flow cytometry. The 4, 12 and 24-week groups were implanted with non-labelled cells and compared with saline-injected controls for healing. RESULTS: The MSCs displayed no reduced viability in vitro to an uptake of 20.0 ± 4.6 pg MIONs per cell, which was detectable by MRI at minimal density of ~ 3 × 104 cells. Treated limbs indicated cellular distribution throughout the tendon synovial sheath but restricted to the synovial tissues, with no MSCs detected in the tendon or surgical lesion. The lesion was associated with negligible morbidity with minimal inflammation post surgery. Evaluation of both treated and control lesions showed no evidence of healing of the lesion at 4, 12 and 24 weeks on gross and histological examination. CONCLUSIONS: Unlike other laboratory animal models of tendon injury, this novel model mimics the failed tendon healing seen clinically intra-synovially. Importantly, however, implanted stem cells exhibited homing to synovium niches where they survived for at least 14 days. This phenomenon could be utilised in the development of novel physical or biological approaches to enhance localisation of cells in augmenting intra-synovial tendon repair.

Mineralization of the Equine Palmar/Plantar Annular Ligament Treated by Surgical Resection.

OBJECTIVE: To document the clinical presentation, diagnosis, and surgical treatment of mineralization of the equine palmar/plantar annular ligament (PAL). STUDY DESIGN: Retrospective study. ANIMALS: Ponies (n=7). METHODS: Case records from 2 referral hospitals were examined to identify cases with lameness associated with PAL mineralization treated surgically. Follow-up information was obtained from the owners by telephone questionnaire. RESULTS: Duration of lameness before referral ranged from 5 weeks to 6 months, and degree of lameness from grade 1 to 5 out of 10. In 3 cases, records noted obvious pain when pressure was applied over the PAL. Pain resulting in lameness was localized to this area and all cases were treated surgically, although the extent of resected tissue varied among cases. Histological examination of resected tissue (4 cases) revealed fibrocartilaginous and/or osseous metaplasia. Following surgery, 6 of the 7 ponies became sound. CONCLUSION: Based on this limited case series, surgical treatment for mineralization of the PAL offers a favorable success rate without severe complications where conservative methods have failed.

Modulation of mesenchymal stem cell genotype and phenotype by extracellular matrix proteins.

AIM: To investigate the effect of extracellular matrix (ECM) proteins on characteristics of mesenchymal stem cells (MSCs) and tendon-derived cells (TDCs). MATERIALS AND METHODS: MSCs and TDCs, cultured in a monolayer (2D) or hydrogels (3D), with or without ECM protein supplementation, and on a non-viable native tendon (NNT) matrix were assayed for adhesion, proliferation, gene expression, and integrin expression. RESULTS: MSCs exhibited a fibroblastic, spindle-shaped morphology on 2D matrices except in the presence of fibronectin. In 3D matrices, MSCs displayed a rounded phenotype except when cultured on NNTs where cells aligned along the collagen fibrils but, unlike TDCs, did not form inter-cellular cytoplasmic processes. MSC proliferation was significantly (p < 0.01) increased by collagen type I in 2D culture and fibronectin in 3D culture. TDC proliferation was unaffected by substrata. MSCs and TDCs differentially expressed α2 integrin. Adhesion to substrata was reduced by RGD-blocking peptide and ß1 integrin antibody. The presence of collagen I or fibronectin upregulated MSC expression of collagen type I and collagen type III, COMP, decorin, osteopontin, and fibronectin. CONCLUSIONS: The morphology, gene expression, and adhesion of both MSCs and TDCs are sensitive to the presence of specific ECM components. Interaction with the ECM is, therefore, likely to affect the mechanism of action of MSCs in vitro and may contribute to phenotypic modulation in vivo.

Immunophenotypic characterization of ovine mesenchymal stem cells.

The clinical potential of multipotent mesenchymal stem cells (MSCs) has led to the essential development of analytical tools such as antibodies against membrane-bound proteins for the immunophenotypic characterization of human and rodent cells. Such tools are frequently lacking for emerging large animal models like the sheep that have greater relevance for the study of human musculoskeletal diseases. The present study identified a set of commercial nonspecies specific monoclonal antibodies for the immunophenotypic characterization of ovine MSCs. A protocol combining the less destructive proteolytic activity of accutase and EDTA was initially developed for the detachment of cells from plastic with minimum loss of cell surface antigens. A range of commercially available antibodies against human or rodent MSC antigens were then tested in single and multistain-based assays for their cross-reactivity to bone marrow derived ovine MSCs. Antibody clones cross-reactive to ovine CD73 (96.9% ± 5.9), CD90 (99.6% ± 0.3), CD105 (99.1 ± 1.5), CD271 (97.7 ± 2.0), and MHC1 (94.0% ± 7.2) antigens were identified using previously reported CD29, CD44, and CD166 as positive controls. Multistaining analysis indicated the colocalization of these antigens on MSCs. Furthermore, antibody clones identified to cross-react against white blood cell antigens exhibited either negative (CD117 (0.1% ± 0.1)) or low (MHCII (10.5% ± 16.0); CD31 (14.6% ± 4.2), and CD45 (39.4% ± 31.8)) cross-reactivity with ovine MSCs. The validation of these antibody clones to sheep MSC antigens is essential for studies utilizing this large animal model for stem cell-based therapies. © 2016 International Society for Advancement of Cytometry.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (<10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers. This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

Changes in synovial fluid biomarker concentrations following arthroscopic surgery in horses with osteochondritis dissecans of the distal intermediate ridge of the tibia.

OBJECTIVE: To quantify concentrations of cartilage oligomeric matrix protein (COMP) and fibromodulin in synovial fluid from the tarsocrural joints (TCJs) of horses with osteochondritis dissecans (OCD) of the distal intermediate ridge of the tibia and determine whether concentrations would change following arthroscopic removal of osteochondral fragments. ANIMALS: 115 client-owned horses with OCD of the TCJ and 29 control horses euthanized for unrelated reasons. PROCEDURES: COMP and fibromodulin concentrations were measured in synovial fluid from the TCJs of the affected horses before and after osteochondral fragments were removed arthroscopically and in synovial fluid from the TCJs of the control horses after euthanasia. Synovial biopsy specimens from the TCJs of affected and control horses were examined histologically for evidence of inflammation. RESULTS: Synovial fluid COMP and fibromodulin concentrations prior to surgery in horses with OCD were not significantly different from concentrations in control horses. Fibromodulin, but not COMP, concentration in horses with OCD was significantly decreased after surgery, compared with the concentration before surgery. Fibromodulin concentration was significantly correlated with joint effusion score but not with lameness score or results of a flexion test and was correlated with histologic score for number of synoviocytes on the surface of the synovium but not with score for degree of infiltration of inflammatory cells in the synovium. Synovial fluid COMP concentration was not significantly correlated with clinical or histologic findings. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that fibromodulin, but not COMP, could potentially be a biomarker of joint inflammation in horses with OCD of the TCJ.

Mesenchymal stem cells modulate release of matrix proteins from tendon surfaces in vitro: a potential beneficial therapeutic effect.

AIM: Injury of tendons contained within a synovial environment, such as joint, bursa or tendon sheath, frequently fails to heal and releases matrix proteins into the synovial fluid, driving inflammation. This study investigated the effectiveness of cells to seal tendon surfaces and provoke matrix synthesis as a possible effective injectable therapy. MATERIALS & METHODS: Equine flexor tendon explants were cultured overnight in suspensions of bone marrow and synovium-derived mesenchymal stems cells and, as controls, two sources of fibroblasts, derived from tendon and skin, which adhered to the explants. Release of the most abundant tendon extracellular matrix proteins into the media was assayed, along with specific matrix proteins synthesis by real-time PCR. RESULTS: Release of extracellular matrix proteins was influenced by the coating cell type. Fibroblasts from skin and tendon appeared less capable of preventing the release of matrix proteins than mesenchymal stems cells. CONCLUSION: The source of cell is an important consideration for cell therapy.

Distribution of injected technetium(99m)-labeled mesenchymal stem cells in horses with naturally occurring tendinopathy.

This study aimed to investigate immediate cell survival and distribution following different administration routes of mesenchymal stem cells (MSCs) into naturally occurring tendon injuries. Ten million MSCs, labeled with technetium-99m hexamethylpropyleneamine oxime, were implanted into 13 horses with naturally occurring tendon or ligament injuries intra-lesionally, intravenously and by regional perfusion, and traced for up to 48 h using planar gamma scintigraphy. Labeling efficiencies varied between 1.8% and 18.5% (mean 9.3%). Cells were retained in the damaged area after intra-lesional administration but only 24% of cells were still present within the tendon after 24 h. After intravenous injection, cells largely distributed to the lung fields, with no detectable cells in the tendon lesions. Significant labeling of the tendon lesions was observed in 11/12 horses following regional perfusion but at a lower level to intra-lesional injection. The highest cell numbers were retained after intra-lesional injection, although with considerable cell loss, while regional perfusion may be a viable alternative for MSC delivery. Cells did not "home" to damaged tendon in large numbers after intravenous administration. Cells were detected in the lungs most frequently after intravascular administration, although with no adverse effects. Low cell retention has important implications for designing effective clinical therapies for human clinical use.

Outcome after lacerations of the superficial and deep digital flexor tendons, suspensory ligament and/or distal sesamoidean ligaments in 106 horses.

OBJECTIVE: To report outcome after the surgical treatment of lacerations of the superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT), suspensory ligament (SL), and/or distal sesamoidean ligaments (DSL) in horses. STUDY DESIGN: Case series. ANIMALS: Horses (n=106) with lacerations of the SDFT, DDFT, SL, and/or DSL. METHODS: Medical records (1988-2002) were reviewed for signalment, limb and tendon/ligament involvement (location and extent of injury, tendon sheath involvement), method of repair, and outcome. RESULTS: The median age of horses was 7 years and the follow-up time ranged from 1 to 10 years. Fifty-five percent of horses returned to their previous level of performance, 27% to a lower level, and 18% were euthanatized. Multivariate statistical analysis demonstrated that the number of structures transected had the most significant influence on outcome. No significant association was detected between outcome and tendon sheath involvement, tendon suturing, casting, or limb affected. Fetlock hyperextension was the most significant complication. CONCLUSIONS: A high survival rate can be expected after SDFT, DDFT, SL, and/or DSL lacerations in horses, but only 55% of affected horses returned to their previous activity level. The number of structures affected was the major factor determining whether horses returned to an equal level of performance.

Increased cartilage oligomeric matrix protein concentrations in equine digital flexor tendon sheath synovial fluid predicts intrathecal tendon damage.

OBJECTIVES: To evaluate digital flexor tendon sheath (DFTS) synovial fluid cartilage oligomeric matrix protein (COMP) concentrations as a molecular marker for intrathecal pathology. STUDY DESIGN: Case control study. ANIMALS: Horses (n=46) with DFTS tenosynovitis; 23 fresh cadaver horses. METHODS: DFTS synovial fluid samples were collected from clinical cases with noninfected DFTS tenosynovitis and from control DFTS. Clinical and surgical findings were recorded, and dissection of control limbs was performed to confirm the DFTS to be grossly normal. Synovial fluid COMP was quantified using a homologous competitive inhibition ELISA. RESULTS: Abnormalities were identified tenoscopically: intrathecal tendon/ligament tearing was identified in 37 cases and 9 had other lesions. In control horses, synovial fluid COMP was higher in younger horses. Clinical cases with intrathecal tendon/ligament tearing had higher synovial fluid COMP than either clinical cases with other lesions, or controls. In horses ≥5 years old, the sensitivity and specificity of the assay was high for diagnosing intrathecal tendon/ligament tearing. CONCLUSIONS: COMP concentrations in DFTS synovial fluid were significantly greater than those in normal horses with noninfected tenosynovitis caused by intrathecal tendon/ligament tearing, but not by other lesions.

A preliminary study on the effect of wounding on transforming growth factor-ß1 and cartilage oligomeric matrix protein concentrations in the skin of horses.

OBJECTIVE: To evaluate whether wound type or site influence the production of cartilage oligomeric matrix protein (COMP) and transforming growth factor ß1 (TGF-ß1), and determine if there is a correlation between TGF-ß1and COMP during healing. STUDY DESIGN: Experimental. ANIMALS: Standardbred horses (n=6), 4-8 years old. METHODS: Six, standardized, full-thickness skin wounds (open, straight, and elliptical) were surgically created on the neck (n=3) and metacarpus (3) on each horse. Wounds were randomly allocated to site and side. Tissue samples were collected before creating wounds and on days 7, 14, and 42. COMP concentration (µg/g dry weight of tissue) was determined using a standard competitive ELISA and TGF-ß1 (ng/g dry weight of tissue) was determined using a commercially available sandwich ELISA. RESULTS: COMP concentrations were higher in intact skin on the neck compared with the metacarpus (P=.02). There was no difference in COMP and TGF-ß1 concentrations between the different wound types or sites during healing. There was no correlation between TGF-ß1 and COMP during healing. CONCLUSIONS: Within the limitations of the study design, production of COMP during healing of skin wounds does not appear to be influenced by wound type or anatomic site, nor does it appear to be correlated with TGF-ß1 concentrations.

Mesenchymal stem cell therapy for equine tendinopathy.

Optimal management of tendon overuse injuries in equine and human athletes should avoid the formation of excessive scar tissue, regenerate normal tendon matrix, and reduce re-injury rates. We hypothesized that the implantation of marrow-derived stromal stem cells (BM-MSCs), in far greater numbers than are present normally within tendon tissue, would synthesize a matrix more closely resembling tendon matrix than scar tissue, and hence increase the capacity to return to performance successfully. This article reviews the technique used clinically in the horse and the current outcome data for horses treated by the autologous implantation of BM-MSCs into moderate to severe acute superficial digital flexor tendon (SDFT) injuries. Bone marrow was aspirated from the sternum under standing sedation. The nucleated adherent cell population (containing the BM-MSCs) were isolated and expanded so that, after approximately three weeks, the cells were re-suspended in the supernatant from the bone marrow and implanted into injured SDFT under ultrasonographic guidance. The horses then entered a 48-week rehabilitation period consisting of an ascending exercise regime. By September 2006, 168 racehorses had undergone this regimen. For horses which had returned to full work, 18% had re-injured, which compared favourably to previous studies on conventional management (56% re-injury rate). No adverse effects were noted other than needle tracts visible ultrasonographically. Autologous implantation of mesenchymal stem cells into tendon injuries may therefore improve clinical outcome although definitive proof of efficacy, which is still lacking, will require randomized controlled trials.