F. M. D. Henson

Exploring the Application of Stem Cells in Tendon Repair and Regeneration

PURPOSE To conduct a systematic review of the current evidence for the effects of stem cells on tendon healing in preclinical studies and human studies. METHODS A systematic search of the PubMed, CINAHL (Cumulative Index to Nursing and Allied Health Literature), Cochrane, and Embase databases was performed for stem cells and tendons with their associated terminology. Data validity was assessed, and data were collected on the outcomes of trials. RESULTS A total of 27 preclinical studies and 5 clinical studies met the inclusion criteria. Preclinical studies have shown that stem cells are able to survive and differentiate into tendon cells when placed into a new tendon environment, leading to regeneration and biomechanical benefit to the tendon. Studies have been reported showing that stem cell therapy can be enhanced by molecular signaling adjunct, mechanical stimulation of cells, and the use of augmentation delivery devices. Studies have also shown alternatives to the standard method of bone marrow-derived mesenchymal stem cell therapy. Of the 5 human studies, only 1 was a randomized controlled trial, which showed that skin-derived tendon cells had a greater clinical benefit than autologous plasma. One cohort study showed the benefit of stem cells in rotator cuff tears and another in lateral epicondylitis. Two of the human studies showed how stem cells were successfully extracted from the humerus and, when tagged with insulin, became tendon cells. CONCLUSIONS The current evidence shows that stem cells can have a positive effect on tendon healing. This is most likely because stem cells have regeneration potential, producing tissue that is similar to the preinjury state, but the results can be variable. The use of adjuncts such as molecular signaling, mechanical stimulation, and augmentation devices can potentially enhance stem cell therapy. Initial clinical trials are promising, with adjuncts for stem cell therapy in development.

Equine dyschondroplasia (osteochondrosis)—Histological findings and type VI collagen localization

This study describes (1) the histological appearance of dyschondroplasia, the primary lesion of osteochondrosis, in articular cartilage of the horse and (2) the localization of type VI collagen which is an important constituent of the extracellular matrix (ECM). Dyschondroplastic cartilage was identified on the basis of the presence of cartilage cores (i.e., cartilage extending into the subchondral bone) and confirmed with subsequent histological examination. Full-thickness cartilage samples from 57 horses were collected and paraffin embedded. Histological examination was used to examine the normal architecture of equine growth cartilage and to determine the presence of various pathological changes in dyschondroplastic lesions. Immunolocalization was used to identify type VI collagen in normal and dyschondroplastic lesions. The abnormalities observed in the dyschondroplastic cartilage fell into two groups. In Group A (n = 18) the lesions were associated with a disruption in the normal sequential transition of the chondrocytes through proliferation and maturation resulting in an accumulation of large numbers of small, rounded chondrocytes. A decrease in type VI collagen immunoreactivity compared with normal animals was detected except around chondrocyte clusters. Group B lesions (n = 9) were characterized by an alteration in the staining pattern of the mineralized cartilage and underlying bone. In these lesions type VI collagen immunoreactivity was increased. In both groups the presence of retained blood vessels, chondrocyte clusters, chondronecrosis and fissure formation was detected. These two histologically-distinct groups suggest that equine dyschondroplasia may be comprised of different pathological entities and that it is associated with alterations in the pattern of distribution of an ECM protein.

Radiographic retrospective study of the caudal cervical articular process joints in the horse.

REASONS FOR PERFORMING STUDY Arthropathy of the caudal cervical articular process joints (APJs) in the horse is documented as a cause of ataxia and paresis secondary to spinal cord compression. Enlargement of the caudal APJs is reported to increase with age, but there are no known associations of any other factors. No association of the degree of APJ enlargement with neurological signs seen has been documented. This study investigated the associations of cervical APJ enlargement at the C5-C6 and C6-C7 articulations with case subject details (breed, age, sex, usage) and clinical signs. OBJECTIVES To ascertain if there are of any associations between: the subject details and enlargement of the caudal cervical APJs; and the degree of APJ enlargement and the presence and type of clinical signs. HYPOTHESES There would be an effect of age, breed and usage on APJ grade, with no effect of sex. Association between grade and clinical signs seen was also investigated. MATERIALS AND METHODS The radiographs of 122 horses qualified for inclusion. Horses were excluded if they were known to have a neck lesion cranial to C5-C6, or if the radiographs were rotated or of poor quality. In order to standardise the interpretation of APJ enlargement, a novel grading system was developed and used. RESULTS An association was found between age and APJ grade at C5-C6 but not C6-C7. There was no association between grade, breed, sex and usage, or clinical signs seen. Data also showed a trend for increasing enlargement the more caudal the APJ. CONCLUSION AND POTENTIAL RELEVANCE The data in this study support that the size of the caudal cervical APJ at the level of C5-C6, appear to increase with age, but this enlargement may not be significant. Enlargement cannot be associated with breed, sex or discipline of the horse at present, and specific grades and therefore degree of enlargement, cannot necessarily be assumed to be the cause of neurological deficits.

Treatment of periocular and non-ocular sarcoids in 18 horses by interstitial brachytherapy with iridium-192

Treatment of the equine sarcoid has posed a significant challenge to clinicians for years and many different methods have been tried with varying success, including ionising radiation. The aim of this study was to review the efficacy of iridium-192 interstitial brachytherapy for the treatment of eight periocular sarcoids and 15 non-ocular sarcoids on 18 horses. All the periocular sarcoids and 13 of the 15 non-ocular sarcoids were treated successfully.

Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC’s were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

Expression of types II, VI and X collagen in equine growth cartilage during development

The synthesis and expression of collagen types II, VI and X were investigated in growth cartilage selected from a group of 31 horses and ponies in the age range 157 days of gestation to 12 years. Collagen isolation, immunolocalisation and in situ hybridisation techniques were used in order to provide information on the pattern of synthesis of these 3 collagens during endochondral ossification in normal horses. Type II collagen immunoreactivity and mRNA expression was found in each of the 3 zones of growth cartilage chondrocytes in all samples studied, whereas the localisation of both collagen types VI and X varied during cartilage development Type VI collagen in the fetus was present only in the resting and upper proliferative zones and around the cartilage canal blood vessels in both articular/epiphyseal and metaphyseal cartilage, whereas in animals age >2 years it was present throughout all the cartilage studied. Type X collagen immunoreactivity and mRNA expression was detected only in the late hypertrophic zone in articular/epiphyseal cartilage in animals age <6 months and in metaphyseal cartilage in animals <12 months. These results demonstrated the presence of collagen types VI and X in equine cartilage for the first time. In addition, the pattern of expression of type II mRNA in the cartilage has been established and type VI and X collagens have been shown to differ in their expression during development of the skeleton.

Effect of a solution of hyaluronic acid- chondroitin sulfate-N-acetyl glucosamine on the repair response of cartilage to single-impact load damage

OBJECTIVE To investigate effects of 1% hyaluronic acid-chondroitin sulfate-N-acetyl glucosamine (HCNAG) on the damage repair response in equine articular cartilage. SAMPLE Articular cartilage from 9 clinically normal adult horses. PROCEDURES Full-thickness cartilage disks were harvested from the third metacarpal bone. Cartilage was single-impact loaded (SIL) with 0.175 J at 0.7 m/s and cultured in DMEM plus 1 % (vol/vol) HCNAG or fibroblastic growth factor (FGF)-2 (50 ng/mL). Histologic and immunohistochemical techniques were used to identify tissue architecture and apoptotic cells and to immunolocalize type I and II collagen and proliferating nuclear cell antigen (PCNA). RESULTS Type II collagen immunoreactivity increased in SIL cartilage, compared with control samples. At days 14 and 28 (day 0 = initiation of culture), control samples had significantly fewer repair cells than did other treatment groups. In control samples and SIL + HCNAG, there was a significant decrease in apoptotic cell number, compared with results for SIL and SIL + FGF-2 samples. At days 14 and 28, there was a significant increase in chondrocytes stained positive for PCNA in the control samples. CONCLUSIONS AND CLINICAL RELEVANCE 1% HCNAG significantly affected apoptotic and repair cell numbers in an SIL damage-repair technique in adult equine articular cartilage. However, HCNAG had no effect on the number of PCNA-positive chondrocytes or on type II collagen immunohistochemical results. The inclusion of 1% HCNAG in lavage solutions administered after arthroscopy may be beneficial to cartilage health by increasing the number of repair cells and decreasing the number of apoptotic cells.

Dynamic compressive strain inhibits nitric oxide synthesis by equine chondrocytes isolated from different areas of the cartilage surface

REASONS FOR PERFORMING STUDY Chondrocytes within articular cartilage respond to the mechanical stresses associated with normal joint loading via a series of signalling pathways. Specific biomolecules, such as nitric oxide (NO), have been implicated in these mechanotransduction processes. It has been shown that the synthesis of NO can be inhibited by dynamic compressive strain of chondrocytes in vitro which, in turn, leads to an up-regulation of specific metabolic parameters. HYPOTHESIS Chondrocytes isolated from different joint locations and seeded in agarose constructs respond in a distinct manner to the application of dynamic compression. METHODS Chondrocytes were isolated separately from the equine patella groove and the femoral condyle, representing high loaded areas (HLA) and low loaded areas (LLA), respectively, of 6 specimens of different ages. The cells were seeded in agarose constructs and cultured either in an unstrained state or strained under dynamic loading at 1 Hz for 48 h. The synthesis of nitric oxide (NO), proteoglycan synthesis and chondrocyte proliferation were assessed. RESULTS Equine chondrocytes were found to synthesise significant basal levels of NO, regardless of topographical origin or age of tissue. Marked differences in both proteoglycan synthesis and cell proliferation were, however, revealed between the 2 chondrocyte subpopulations. Dynamic compression inhibited NO synthesis but significant alterations in proteoglycan synthesis and cell proliferation were apparent in a minority of cases. CONCLUSIONS AND POTENTIAL RELEVANCE The differential response of the subpopulations of chondrocytes derived from the HLA and LLA provides a potential mechanism which enables the biomechanical demands of differing joint regions to be maintained.

Equine carpal articular cartilage fibronectin distribution associated with training, joint location and cartilage deterioration.

Processes involved in equine carpal osteochondral injury have not been established. In other species, fibronectin appears important in chondrocyte-matrix interactions, and levels are increased in osteoarthritis. This investigation aimed to (a) describe fibronectin immunoreactivity in the middle carpal joint of 2-year-old Thoroughbreds, (b) determine topographical variations, (c) compare strenuously trained (Group 1) or gently exercised horses (Group 2) and (d) describe sites with early osteoarthritis. Group 1 (n = 6) underwent a 19 week high intensity treadmill training programme. Group 2 (n = 6) underwent 40 min walking until euthanasia. Dorsal and palmar sites on radial, intermediate and third carpal articular surfaces were prepared. Immunohistochemistry was performed using a biotin-streptavidin/peroxidase method. Cross-reactivity of rabbit antihuman fibronectin antiserum with equine fibronectin was confirmed using Western blotting. Results showed: (a) fibronectin was present primarily in pericellular and interterritorial matrix locations, (b) dorsal sites had zonal immunoreactivity compared to palmar sites, (c) Group 1 dorsal radial carpal cartilage had increased superficial staining compared to Group 2 and (d) fibrillated cartilage showed increased intracellular and local matrical immunoreactivity (superficial zone). These findings suggest topographical and exercise-related variations in fibronectin distribution, and indicate equine fibronectin is localised at sites of cartilage degeneration and released into the matrix by chondrocytes in the local area.

The Augmentation of a Collagen/Glycosaminoglycan Biphasic Osteochondral Scaffold with Platelet-Rich Plasma and Concentrated Bone Marrow Aspirate for Osteochondral Defect Repair in Sheep: A Pilot Study.

Objective: This study investigates the combination of platelet-rich plasma (PRP) or concentrated bone marrow aspirate (CBMA) with a biphasic collagen/glycosaminoglycan (GAG) osteochondral scaffold for the treatment of osteochondral defects in sheep. Design: Acute osteochondral defects were created in the medial femoral condyle (MFC) and the lateral trochlea sulcus (LTS) of 24 sheep (n = 6). Defects were left empty or filled with a 6 × 6-mm scaffold, either on its own or in combination with PRP or CBMA. Outcome measures at 6 months included mechanical testing, International Cartilage Repair Society (ICRS) repair score, modified O’Driscoll histology score, qualitative histology, and immunohistochemistry for type I, II, and VI collagen. Results: No differences in mechanical properties, ICRS repair score, or modified O’Driscoll score were detected between the 4 groups. However, qualitative assessments of the histological architecture, Safranin O content, and collagen immunohistochemistry indicated that in the PRP/scaffold groups, there was a more hyaline cartilage–like tissue repair. In addition, the addition of CBMA and PRP to the scaffold reduced cyst formation in the subchondral bone of healed lesions. Conclusion: There was more hyaline cartilage–like tissue formed in the PRP/scaffold group and less subchondral cystic lesion formation in the CBMA and PRP/scaffold groups, although there were no quantitative differences in the repair tissue formed.