One-stage surgical case management of a two-year-old Arabian horse affected by male-pseudo hermaphroditism.

A two-year-old Arabian horse presented for abnormal external genitalia and dangerous stallion-like behavior was diagnosed with disorder of sexual development (DSD), also known as intersex/hermaphroditism. Standing 1-stage surgical procedure performed under sedation, and local anesthesia to concurrently eliminate stallion-like behavior, risk of neoplastic transformation of intraabdominal gonads, and to replace ambiguous external genital with a functional, and cosmetically more acceptable anatomy. Step-1) Laparoscopic abdominal exploration and gonadectomy; Step-2) Rudimentary penis resection and perineal urethrostomy. The horse tolerated surgery well (combined surgery time 185 min) with no complications. At macroscopic examination of the gonads, they resembled hypoplastic testis-like tissues. Microscopic examination confirmed presence of seminiferous tubules, Leydig and Sertoli/granulosa cells. Cytogenetic evaluation revealed a 64,XX karyotype, SRY-negative. The stallion-like behavior subsided within days post-operatively. Long-term follow-up revealed the genitoplasty site healed without urine scalding or urethral stricture. The owner satisfaction was excellent and the horse could be used post-surgery as an athlete.

Tissue engineering scaled-up, anatomically shaped osteochondral constructs for joint resurfacing.

Arthroplasty is currently the only surgical procedure available to restore joint function following articular cartilage and bone degeneration associated with diseases such as osteoarthritis (OA). A potential alternative to this procedure would be to tissue-engineer a biological implant and use it to replace the entire diseased joint. The objective of this study was therefore to tissue-engineer a scaled-up, anatomically shaped, osteochondral construct suitable for partial or total resurfacing of a diseased joint. To this end it was first demonstrated that a bone marrow derived mesenchymal stem cell seeded alginate hydrogel could support endochondral bone formation in vivo within the osseous component of an osteochondral construct, and furthermore, that a phenotypically stable layer of articular cartilage could be engineered over this bony tissue using a co-culture of chondrocytes and mesenchymal stem cells. Co-culture was found to enhance the in vitro development of the chondral phase of the engineered graft and to dramatically reduce its mineralisation in vivo. In the final part of the study, tissue-engineered grafts (~ 2 cm diameter) mimicking the geometry of medial femorotibial joint prostheses were generated using laser scanning and rapid prototyped moulds. After 8 weeks in vivo, a layer of cartilage remained on the surface of these scaled-up engineered implants, with evidence of mineralisation and bone development in the underlying osseous region of the graft. These findings open up the possibility of a tissue-engineered treatment option for diseases such as OA.

Cell-matrix interactions regulate mesenchymal stem cell response to hydrostatic pressure.

Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the response of MSCs to hydrostatic pressure will depend on the biomaterial within which the cells are encapsulated. Bone-marrow-derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4 h per day, 5 days per week for 3 weeks) in the presence of either 1 or 10 ng ml(-1) of TGF-ß3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulfated glycosaminoglycan (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure, which plays a key role in regulating the chondrogenic differentiation of MSCs.

Composition-function relations of cartilaginous tissues engineered from chondrocytes and mesenchymal stem cells isolated from bone marrow and infrapatellar fat pad.

The objective of this study was to determine the functional properties of cartilaginous tissues generated by porcine MSCs isolated from different tissue sources, and to compare these properties to those derived from chondrocytes (CCs). MSCs were isolated from bone marrow (BM) and infrapatellar fat pad (FP), while CCs were harvested from the articular surface of the femoro-patellar joint. Culture-expanded CCs and MSCs were encapsulated in agarose hydrogels and cultured in the presence of TGFß3. Samples were analysed biomechanically, biochemically and histologically at days 0, 21 and 42. After 42 days in free swelling culture, mean GAG content was 1.50% w/w in CC-seeded constructs, compared to 0.95% w/w in FP- and 0.43% w/w in BM-seeded constructs. Total collagen accumulation was highest in FP constructs. DNA content increased with time for all the groups. The mechanical functionality of cartilaginous tissues engineered using CCs was superior to that generated from either source of MSCs. Differences were also observed in the spatial distribution of matrix components in tissues engineered using CCs and MSCs, which appears to have a strong influence on the apparent mechanical properties of the constructs. Therefore, while functional cartilaginous tissues can be engineered using MSCs isolated from different sources, the spatial composition of these tissues is unlike that generated using chondrocytes, suggesting that MSCs and chondrocytes respond differently to the regulatory factors present within developing cartilaginous constructs.

Oxygen tension differentially regulates the functional properties of cartilaginous tissues engineered from infrapatellar fat pad derived MSCs and articular chondrocytes.

BACKGROUND: For current tissue engineering or regenerative medicine strategies, chondrocyte (CC)- or mesenchymal stem cell (MSC)-seeded constructs are typically cultured in normoxic conditions (20% oxygen). However, within the knee joint capsule a lower oxygen tension exists. OBJECTIVE: The objective of this study was to investigate how CCs and infrapatellar fad pad derived MSCs will respond to a low oxygen (5%) environment in 3D agarose culture. Our hypothesis was that culture in a low oxygen environment (5%) will enhance the functional properties of cartilaginous tissues engineered using both cell sources. EXPERIMENTAL DESIGN: Cell-encapsulated agarose hydrogel constructs (seeded with CCs or infrapatellar fat pad (IFP) derived MSCs) were prepared and cultured in a chemically defined serum-free medium in the presence (CCs and MSCs) or absence (CCs only) of transforming growth factor-beta3 (TGF-ß3) in normoxic (20%) or low oxygen (5%) conditions for 42 days. Constructs were assessed at days 0, 21 and 42 in terms of mechanical properties, biochemical content and histologically. RESULTS: Low oxygen tension (5%) was observed to promote extracellular matrix (ECM) production by CCs cultured in the absence of TGF-ß3, but was inhibitory in the presence of TGF-ß3. In contrast, a low oxygen tension enhanced chondrogenesis of IFP constructs in the presence of TGF-ß3, leading to superior mechanical functionality compared to CCs cultured in identical conditions. CONCLUSIONS: Extrapolating the results of this study to the in vivo setting, it would appear that joint fat pad derived MSCs may possess a superior potential to generate a functional repair tissue in low oxygen tensions. However, in the context of in vitro cartilage tissue engineering, CCs maintained in normoxic conditions in the presence of TGF-ß3 generate the most mechanically functional tissue.

Chondrogenesis and integration of mesenchymal stem cells within an in vitro cartilage defect repair model.

Integration of repair tissue is a key indicator of the long-term success of cell-based therapies for cartilage repair. The objective of this study was to compare the in vitro chondrogenic differentiation and integration of agarose hydrogels seeded with either chondrocytes or bone marrow-derived mesenchymal stem cells (MSCs) in defects created in cartilage explants. Chondrocytes and MSCs were isolated from porcine donors, suspended in 2% agarose and then injected into cylindrical defects within the explants. These constructs were maintained in a chemically defined medium supplemented with 10 ng/mL of TGF-beta3. Cartilage integration was assessed by histology and mechanical push-out tests. After 6 weeks in culture, chondrocyte-seeded constructs demonstrated a higher integration strength (64.4 +/- 8.3 kPa) compared to MSC-seeded constructs (22.7 +/- 5.9 kPa). Glycosaminoglycan (GAG) (1.27 +/- 0.3 vs. 0.19 +/- 0.03 kPa) and collagen (0.31 +/- 0.08 vs. 0.09 +/- 0.01 kPa) accumulation in chondrocyte-seeded constructs was greater than that measured in the MSC-seeded group. The GAG, collagen, and DNA content of both chondrocyte- and MSC-seeded hydrogels cultured in cartilage explants was significantly lower than control constructs cultured in free swelling conditions. The results of this study suggest that the explant model may constitute a more rigorous in vitro test to assess MSC therapies for cartilage defect repair.

Evidence to suggest that cathepsin K degrades articular cartilage in naturally occurring equine osteoarthritis.

OBJECTIVE: The mechanisms leading to degeneration of articular cartilage in osteoarthritis (OA) are complex and not yet fully understood. Cathepsin K (CK) is a cysteine protease which can also cleave the triple helix of type II collagen. This exposes a neoepitope that can now be identified by specific antibodies. The aim of this study was to obtain evidence suggesting a role for CK in naturally occurring equine OA in both lesional and peri-lesional regions. METHODS: Articular cartilages (n=12 horses; 5 healthy, 7 OA) were harvested from animals postmortem. A gross macroscopic examination, histologic (Safranin O-Fast Green and Picrosirius red staining) and immunohistochemical evaluation were performed. Samples were divided into normal appearing cartilage, peri-lesional and lesional cartilage. Cartilage degradation in the samples was graded histologically and immunohistochemically. CK and possible CK cleavage were detected immunohistochemically with specific anti-protein and anti-neoepitope antibodies, respectively. A comparison of CK neoepitope (C2K) production with the collagenase-generated neoepitope produced by matrix metalloproteinases (MMP)-1, 8 and 13 (C2C) was also assessed immunohistochemically. RESULTS: CK and CK cleavage were significantly more abundant in OA cartilage (both peri-lesional and lesional) when compared to remote cartilage within the sample joint or cartilage from healthy joints. The immunohistochemical pattern observed for CK degradation (C2K) was similar to that of collagenase degradation (C2C). Macroscopic cartilage changes and histologic findings were significantly correlated with immunohistochemistry results. CONCLUSION: The data generated suggests that CK may be involved in cartilage collagen degradation in naturally occurring osteoarthritis.

Joint inflammation increases glucosamine levels attained in synovial fluid following oral administration of glucosamine hydrochloride.

OBJECTIVE: To compare synovial glucosamine levels in normal and inflamed equine joints following oral glucosamine administration and to determine whether single dose administration alters standard synovial parameters of inflammation. METHODS: Eight adult horses were studied. On weeks 1 and 2, all horses received 20mg/kg glucosamine hydrochloride by nasogastric (NG) intubation or intravenous injection. On weeks 3 and 4, 12h after injection of both radiocarpal joints with 0.25 ng Escherichia coli lipopolysaccharide (LPS) to induce inflammation, glucosamine hydrochloride or a placebo was administered by NG intubation. Plasma samples were collected at baseline and 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid (SF) samples were collected within 48 h before dosing and 1, 6 and 12h post-dosing. Glucosamine was analyzed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). Clinicopathological evaluation of SF parameters included white blood cell (WBC) count and total protein (TP) analyses. RESULTS: No significant differences between groups were observed in SF baseline levels of WBC and TP at any stage of the study. SF WBC and TP significantly increased following IA LPS. The mean (+/-SD) maximal SF glucosamine levels (422.3+/-244.8 ng/mL) were significantly higher (>fourfold) in inflamed joints when compared to healthy joints (92.7+/-34.9 ng/mL). Glucosamine did not have any effect on standard SF parameters of inflammation. CONCLUSION: Synovial inflammation leads to significantly higher synovial glucosamine concentrations compared to levels attained in healthy joints following oral administration of glucosamine hydrochloride. Whether these higher levels are translated into a therapeutic effect on the joint tissues remains to be elucidated.

Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells.

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-beta3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5+/-0.21%w/w vs. 0.94+/-0.03%w/w) and annulus (1.09+/-0.09%w/w vs. 0.59+/-0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.

Comparison of pharmacokinetics of glucosamine and synovial fluid levels following administration of glucosamine sulphate or glucosamine hydrochloride.

OBJECTIVE: To compare the pharmacokinetics of glucosamine and the synovial fluid levels attained following treatment with glucosamine sulphate or glucosamine hydrochloride in a large animal model at clinically relevant doses. METHODS: Eight adult female horses were used. Crystalline glucosamine sulphate (Dona) or glucosamine hydrochloride was administered at a dose of 20 mg/kg by either intravenous (i.v.) injection or nasogastric (n.g.) intubation. Plasma samples were collected before dosing and at 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid samples were collected from the radiocarpal joints within 48 h before dosing and at 1, 6 and 12 h post-dosing. Glucosamine was assayed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). RESULTS: Plasma concentrations reached approximately 50 microg/mL after i.v. injection and approximately 1 microg/mL after n.g. administration of both types of glucosamine. The median oral bioavailability was 9.4% for glucosamine sulphate and 6.1% for glucosamine hydrochloride. Synovial fluid concentrations were significantly higher at 1 and 6 h following oral treatment with glucosamine sulphate compared to glucosamine hydrochloride. Twelve hours following oral administration, glucosamine levels in the plasma and the synovial fluid were still significantly higher than baseline for the glucosamine sulphate preparation, but not for the hydrochloride preparation. CONCLUSION: Following oral administration of a clinically recommended dose of glucosamine sulphate (Dona), significantly higher synovial fluid concentrations of glucosamine are attained, when compared to an equivalent dose of glucosamine hydrochloride. Whether this difference is translated into a therapeutic effect on the joint tissues remains to be elucidated.