Treatment Effects of Intra-Articular Allogenic Mesenchymal Stem Cell Secretome in an Equine Model of Joint Inflammation.

Background: Allogenic mesenchymal stem cell (MSC) secretome is a novel intra-articular therapeutic that has shown promise in in vitro and small animal models and warrants further investigation. Objectives: To investigate if intra-articular allogenic MSC-secretome has anti-inflammatory effects using an equine model of joint inflammation. Study Design: Randomized positively and negatively controlled experimental study. Method: In phase 1, joint inflammation was induced bilaterally in radiocarpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After 2 h, the secretome of INFy and TNFα stimulated allogeneic equine MSCs was injected in one randomly assigned joint, while the contralateral joint was injected with medium (negative control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded, and synovial fluid samples were analyzed for biomarkers (total protein, WBCC; eicosanoid mediators, CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed post-injection hours (PIH 0, 8, 24, 72, and 168 h). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were evaluated. For phase 2, allogeneic MSC-secretome vs. allogeneic equine MSCs (positive control) was tested using a similar methodology. Results: In phase 1, the joint circumference was significantly (p < 0.05) lower in the MSC-secretome treated group compared to the medium control group at PIH 24, and significantly higher peak synovial GAG values were noted at PIH 24 (p < 0.001). In phase 2, no significant differences were noted between the treatment effects of MSC-secretome and MSCs. Main Limitations: This study is a controlled experimental study and therefore cannot fully reflect natural joint disease. In phase 2, two therapeutics are directly compared and there is no negative control. Conclusions: In this model of joint inflammation, intra-articular MSC-secretome injection had some clinical anti-inflammatory effects. An effect on cartilage metabolism, evident as a rise in GAG levels was also noted, although it is unclear whether this could be considered a beneficial or detrimental effect. When directly comparing MSC-secretome to MSCs in this model results were comparable, indicating that MSC-secretome could be a viable off-the-shelf alternative to MSC treatment.

Biomechanical, Morphological, and Biochemical Characteristics of Articular Cartilage of the Ovine Humeral Head.

OBJECTIVE: Shoulder pain is commonly attributed to rotator cuff injury or osteoarthritis. Ovine translational models are used to investigate novel treatments aimed at remedying these conditions to prevent articular cartilage degeneration and subsequent joint degradation. However, topographical properties of articular cartilage in the ovine shoulder are undefined. This study investigates the biomechanical, morphological, and biochemical attributes of healthy ovine humeral head articular cartilage and characterizes topographical variations between surface locations. DESIGN: Ten humeral heads were collected from healthy skeletally mature sheep and each was segregated into 4 quadrants using 16 regions of interest (ROIs) across the articular surface. Articular cartilage of each ROI was analyzed for creep indentation, thickness, and sulfated glycosaminoglycan (sGAG) and collagen quantity. Comparisons of each variable were made between quadrants and between ROIs within each quadrant. RESULTS: Percent creep, thickness, and sGAG content, but not collagen content, were significantly different between humeral head quadrants. Subregion analysis of the ROIs within each surface quadrant revealed differences in all measured variables within at least one quadrant. Percent creep was correlated with sGAG (r = -0.32, P = 0.0001). Collagen content was correlated with percent creep (r = 0.32, P = 0.0009), sGAG (r = -0.19, P = 0.049), and thickness (r = -0.19, P = 0.04). CONCLUSIONS: Topographical variations exist in mechanical, morphologic, and biochemical properties across the articular surface of the ovine humeral head. Recognizing this variability in ovine humeral head cartilage will provide researchers and clinicians with accurate information that could impact study outcomes.

Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics.

BACKGROUND: Umbilical cord (UC) connective tissues contain plastic-adherent, colony forming unit-fibroblasts (CFU-Fs) amenable to culture expansion for potential therapeutic use. Recently, UC-derived allograft products have been made available to practitioners in orthopaedics and other specialties, by companies purporting "stem cell"-based healing. However, such marketing claims conflict with existing regulations for these human tissues, generating questions over the cellular and protein composition of current commercially available UC allograft products. PURPOSE: To evaluate commercial UC allograft products for viable cells, CFU-Fs, and protein makeup. STUDY DESIGN: Descriptive laboratory study. METHODS: Five commercial UC allograft products claiming to contain viable, undescribed "stem cells," 2 obtained from UC blood (UCB) and 3 from UC tissue (UCT), were analyzed. Image-based methods were used to measure cell concentration and viability, a traditional CFU-F assay was used to evaluate in vitro behavior indicative of a connective tissue progenitor cell phenotype often referred to as mesenchymal stem/stromal cells, and quantitative immunoassay arrays were used to measure a combination of cytokines and growth factors. Bone marrow concentrate (BMC) and plasma derived from the blood and bone marrow of middle-aged individuals served as comparative controls for cell culture and protein analyses, respectively. RESULTS: Viable cells were identified within all 5 UC allograft products, with those derived from UCB having greater percentages of living cells (40%-59%) than those from UCT (1%-22%). Compared with autologous BMC (>95% viability and >300 million living cells), no CFU-Fs were observed within any UC allograft product (<15 million living cells). Moreover, a substantial number of proteins, particularly those within UCB allograft products, were undetectable or present at lower concentrations compared with blood and bone marrow plasma controls. Interestingly, several important growth factors and cytokines, including basic fibroblast growth factor, hepatocyte growth factor, interleukin-1 receptor antagonist, and osteoprotegerin, were most prevalent in 1 or more UCT allograft products as compared with blood and bone marrow plasma. CONCLUSION: CFU-Fs, often referred to as stem cells, were not found within any of the commercial UC allograft products analyzed, and clinicians should remain wary of marketing claims stating otherwise. CLINICAL RELEVANCE: Any therapeutic benefit of current UC allograft products in orthopaedic medicine is more likely to be attributed to their protein composition (UCT > UCB) or inclusion of cells without colony forming potential (UCB > UCT).

Mechanical, biochemical, and morphological topography of ovine knee cartilage.

The knee is the most common site for translational cartilage research in sheep, though topographic features of articular cartilage across surfaces are unspecified. We aimed to characterize the mechanical, morphological, and biochemical properties of articular cartilage across ovine knee surfaces and document variations between and within surface locations. Regions of interest (ROIs) were delineated across surfaces of 10 healthy ovine knees. Articular cartilage at each ROI was measured for creep indentation, thickness, and glycosaminoglycan (GAG) and collagen content. Variables were compared between surface locations (trochlea, and lateral [LFC] and medial [MFC] femoral condyles) and between ROIs within each surface location. Correlations between variables were also assessed. Articular surface location had a significant effect on creep (P < .0001), thickness (P < .0001), and collagen (P = .0007), but not GAG (P = .28). Significant differences in percent creep between ROIs were found within the LFC (P < .0001), MFC (P < .0001), and trochlea (P = .0002). Cartilage thickness was different between ROIs within the LFC, MFC, and trochlea (all P < .0001). The LFC (P = .002) and trochlea (P = .01) each had significant differences in GAG between ROIs. Collagen content between ROIs was different within the LFC (P = .0003), MFC (P = .0005), and trochlea (P < .0001). Collagen content was correlated with thickness (r = -.55), percent creep (r = .47), and GAG (r = -.21). Percent creep was correlated with thickness (r = -.64) and GAG (r = -.19). Topographic variations in mechanical, morphological, and biochemical properties exist across knee cartilage surfaces in sheep. Recognition of this variability is important to optimize study protocols and improve accuracy of results.

Adult ovine connective tissue cells resemble mesenchymal stromal cells in their propensity for extensive ex vivo expansion.

Purpose/Aim: Expanded, human connective tissue cells can adopt mesenchymal stromal cell (MSC) properties that are favorable for applications in regenerative medicine. Sheep are used as a large animal model for cell therapies, although for preclinical testing it is important to establish whether ovine cells resemble humans in their tendency to adopt MSC properties. The objective of this study was to investigate whether cells from five ovine connective tissues are MSC-like in their propensity for extensive expansion and immunophenotype.Materials and Methods: Monolayer cultures were established with cells from annulus fibrosus, cartilage, meniscus, tendon, and nucleus pulposus. Bone marrow MSCs were evaluated as a control. Cultures were seeded at 500 cells/cm2, and subcultured every 5 days up to day 20. Flow cytometry was used to evaluate expression of cluster of differentiation (CD) molecules associated with MSCs (29, 44, 166). Colony formation was evaluated using time-lapse imaging of individual cells.Results: By day 20, cumulative population doublings ranged between 22 (chondrocytes) and 27 (MSCs). All cells uniformly expressed CD44 and 73. Expression of CD166 for MSCs was 98-99%, and ranged between 64 and 97% for the other cell types. Time-lapse imaging demonstrated that 58-94% of the cells colonized as indicated by 3 population doublings within 52 hours.Conclusions: Cells from ovine connective tissues resembled MSCs in their propensity for sustained, colony-forming growth and expression of CD molecules. These data supports the potential for preclinical testing of MSC-like connective tissue cells in sheep.

The platelet-rich plasma and mesenchymal stem cell milieu: A review of therapeutic effects on bone healing.

Platelet-rich plasma is autologous plasma that contains concentrated platelets compared to whole blood. It is relatively inexpensive to produce, can be easily isolated from whole blood, and can be administered while the patient is in the operating room. Further, because platelet-rich plasma is an autologous therapy, there is minimal risk for adverse reactions to the patient. Platelet-rich plasma has been used to promote bone regeneration due to its abundance of concentrated growth factors that are essential to wound healing. In this review, we summarize the methods for producing platelet-rich plasma and the history of its use in bone regeneration. We also summarize the growth factor profiles derived from platelet-rich plasma, with emphasis on those factors that play a direct role in promoting bone repair within the local fracture environment. In addition, we discuss the potential advantages of combining platelet-rich plasma with mesenchymal stem cells, a multipotent cell type often obtained from bone marrow or fat, to improve craniofacial and long bone regeneration. We detail what is currently known about how platelet-rich plasma influences mesenchymal stem cells in vitro, and then highlight the clinical outcomes of administering platelet-rich plasma and mesenchymal stem cells as a combination therapy to promote bone regeneration in vivo.

Adult ovine chondrocytes in expansion culture adopt progenitor cell properties that are favorable for cartilage tissue engineering.

Human chondrocytes in expansion culture can become progenitor-like in their ability to proliferate extensively and secrete neocartilage in chondrogenic culture. Sheep are used as a large animal model for cartilage tissue engineering, although for testing progenitor-like chondrocytes it is important that ovine chondrocytes resemble human in the ability to adopt progenitor properties. Here, we investigate whether ovine chondrocytes can adopt progenitor properties as indicated by rapid proliferation in a colony-forming fashion, and high levels of neocartilage secretion in chondrogenic culture. In conditions known to promote expansion of mesenchymal stromal cells, ovine chondrocytes proliferated through approximately 12 population doublings in 10 days. Time-lapse imaging indicated rapid proliferation in a colony-forming pattern. Expanded ovine chondrocytes that were seeded into agarose and cultured in chondrogenic medium accumulated neocartilage over 2 weeks, to a greater extent than primary chondrocytes. These data confirm that ovine chondrocytes resemble human chondrocytes in their ability to acquire progenitor properties that are important for cartilage tissue engineering. Given the broad interest in using progenitor cells to heal connective tissues, next we compared proliferation and trilineage differentiation of ovine chondrocytes, meniscus cells, and tenocytes. Meniscus cells and tenocytes experienced more than 13 population doublings in 10 days. In chondrogenic culture, cartilage matrix accumulation, and gene expression were largely similar among the cell types. All cell types resisted osteogenesis, while expanded tenocytes and meniscal cells were capable of adipogenesis. While ovine connective tissue cells demonstrated limited lineage plasticity, these data support the potential to promote certain progenitor properties with expansion.

Culture Conditions that Support Expansion and Chondrogenesis of Middle-Aged Rat Mesenchymal Stem Cells.

OBJECTIVE: Rats are an early preclinical model for cartilage tissue engineering, and a practical species for investigating the effects of aging. However, rats may be a poor aging model for mesenchymal stem cells (MSCs) based on laboratory reports of a severe decline in chondrogenesis beyond young adulthood. Such testing has not been conducted with MSCs seeded in a scaffold, which can improve the propensity of MSCs to undergo chondrogenesis. Therefore, the objective of this study was to evaluate chondrogenesis of middle-aged rat MSCs encapsulated in agarose. DESIGN: MSCs from 14- to 15-month-old rats were expanded, seeded into agarose, and cultured in chondrogenic medium with or without 5% serum for 15 days. Samples were evaluated for cell viability and cartilaginous extracellular matrix (ECM) accumulation. Experiments were repeated using MSCs from 6-week-old rats. RESULTS: During expansion, middle-aged rat MSCs demonstrated a diminishing proliferation rate that was improved ~2-fold in part by transient exposure to chondrogenic medium. In agarose culture in defined medium, middle-aged rat MSCs accumulated ECM to a much greater extent than negative controls. Serum supplementation improved cell survival ~2-fold, and increased ECM accumulation ~3-fold. Histological analysis indicated that defined medium supported chondrogenesis in a subset of cells, while serum-supplementation increased the frequency of chondrogenic cells. In contrast, young rat MSCs experienced robust chondrogenesis in defined medium that was not improved with serum-supplementation. CONCLUSIONS: These data demonstrate a previously-unreported propensity of middle-aged rat MSCs to undergo chondrogenesis, and the potential of serum to enhance chondrogenesis of aging MSCs.

Can Extracorporeal Shockwave Promote Osteogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells In Vitro?

Both bone marrow-derived mesenchymal stem cells (BMDMSCs) and extracorporeal shockwave (ESW) have shown promise for enhancing fracture repair. If exposure of BMDMSCs to ESW enhances osteogenic differentiation, these therapies may be combined in vivo or used as a method for preconditioning BMDMSCs. The objective of this study was to determine the effect of ESW on the osteogenic ability of equine BMDMSCs. We hypothesized that ESW would promote osteogenesis evidenced by increased gene expression, alkaline phosphatase (ALPL) expression, slide morphologic score, and protein expression. BMDMSCs were evaluated from six horses. BMDMSCs were culture expanded to passage 3, dissociated, then placed in conical tubes. Treatment cells ("shocked") were exposed to 500 pulses at 0.16 mJ/mm2 energy. Cells were then reseeded and grown in either growth medium or osteogenic medium. Cellular proliferation and trilineage potential were determined. Cellular morphology was scored and cells were harvested at 1, 3, 7, 14, and 21 days for rtPCR gene expression of osteogenic markers [osteonectin (ONT), osteocalcin (OCN), ALPL, collagen type 3 (COL3), and runt-related transcription factor 2 (RUNX2)]. Media supernatants were evaluated for secretion of BMP-2, VEGF, TGFß, and PGE2 and cellular lysates were evaluated for ALPL production. There was no difference between the proliferative ability of shocked cells versus unshocked cells in either growth medium or osteogenic medium. ALPL production was greater in shocked cells maintained in osteogenic medium versus unshocked cells in osteogenic medium at day 3 (P < 0.005). Independent of media type, ESW caused a decrease in VEGF and TGFß production at day 3. No significant increases in gene expression were identified by rtPCR. Exposure of BMDMSCs to ESW does not result in negative effects. An initial significant increase in ALPL was detected but no persistent osteogenic effect was observed with cell expansion.

Differential Effects of the Antioxidants N-Acetylcysteine and Pyrrolidine Dithiocarbamate on Mesenchymal Stem Cell Chondrogenesis.

INTRODUCTION: Mesenchymal stem cell (MSC) chondrogenesis is associated with increases in intracellular reactive oxygen species (ROS), which may result in oxidative stress that is detrimental to cartilage regeneration. This study evaluated the ability of the antioxidants N-acetylcysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) to reduce intracellular ROS, and their effect on MSC chondrogenesis and maturation of cartilage-like extracellular matrix. METHODS: Equine bone marrow MSCs were cultured in serum-supplemented chondrogenic medium with or without NAC or PDTC. ROS was quantified in monolayer after 8 and 72 h of culture. MSCs were seeded into agarose, cultured for 15 days, and analyzed for viable cell density, glycosaminoglycan (GAG) and hydroxyproline accumulation, and collagen gene expression. PDTC cultures were evaluated for oxidative damage by protein carbonylation, and mechanical properties via compressive testing. RESULTS: NAC significantly lowered levels of ROS after 8 but not 72 h, and suppressed GAG accumulation (70%). In secondary experiments using serum-free medium, NAC significantly increased levels of ROS at 72 h, and lowered cell viability and extracellular matrix accumulation. PDTC significantly reduced levels of ROS (~ 30%) and protein carbonylation (27%), and enhanced GAG accumulation (20%). However, the compressive modulus for PDTC-treated samples was significantly lower (40%) than controls. Gene expression was largely unaffected by the antioxidants. CONCLUSIONS: NAC demonstrated a limited ability to reduce intracellular ROS in chondrogenic culture, and generally suppressed accumulation of extracellular matrix. Conversely, PDTC was an effective antioxidant that enhanced GAG accumulation, although the concomitant reduction in compressive properties is a significant limitation for cartilage repair.

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self-assembling peptide hydrogel.

Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self-assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self-assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the absence of the chondrogenic cytokine transforming growth factor beta (TGFß). Positive controls consisted of undifferentiated MSCs seeded into agarose and cultured in medium containing TGFß. Three days of preconditioning was sufficient to produce chondrogenic MSCs that accumulated ∼75% more cartilaginous extracellular matrix than positive controls by day 17. However, gene expression of type X collagen was ∼65-fold higher than positive controls, which was attributed to the absence of TGFß. Potential induction of immunogenicity with preconditioning culture was indicated by expression of major histocompatibility complex class II (MHCII), which was nearly absence in undifferentiated MSCs, and ∼7% positive for preconditioned cells. These data demonstrate the potential to generate chondrogenic MSCs with days of self-assembling peptide hydrogel, and the ability to readily recover an individual cell suspension that is suited for injectable therapies. However, continued exposure to TGFß may be necessary to prevent hypertrophy indicated by type X collagen expression, while immunogenicity may be a concern for allogeneic applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1368-1375, 2019.

Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture.

Chondrogenesis of mesenchymal stem cells (MSCs) is induced in culture conditions that have been associated with oxidative stress, although the extent to which the oxidative environment affects differentiation and extracellular matrix (ECM) accumulation is not known. The objectives of this study were to evaluate the oxidative environment during MSCs chondrogenesis in conventional serum-free medium, and the effect of serum-supplementation on intracellular reactive oxygen species (ROS) and chondrogenesis. Young adult equine MSCs were seeded into agarose and cultured in chondrogenic medium, with or without 5% fetal bovine serum (FBS), for up to 15 days. Samples were evaluated for intracellular ROS, the antioxidant glutathione, ECM and gene expression measures of chondrogenesis, and carbonylation as an indicator of oxidative damage. Intracellular ROS increased with time in culture, and was lower in medium supplemented with FBS. Glutathione decreased ∼12-fold during early chondrogenesis (p < 0.0001), and was not affected by FBS (p = 0.25). After 15 days of culture, FBS supplementation increased hydroxyproline accumulation ∼80% (p = 0.0002); otherwise, measures of chondrogenesis were largely unaffected. Protein carbonylation in chondrogenic MSCs cultures was not significantly different between serum-free and FBS cultures (p = 0.72). Supplementation with adult equine serum increased hydroxyproline accumulation by 45% over serum-free culture (p = 0.0006). In conclusion, this study characterized changes in the oxidative environment during MSC chondrogenesis, and suggested that lowering ROS may be an effective approach to increase collagen accumulation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:506-514, 2018.

Growth Factor-Mediated Migration of Bone Marrow Progenitor Cells for Accelerated Scaffold Recruitment.

Tissue engineering approaches using growth factor-functionalized acellular scaffolds to support and guide repair driven by endogenous cells are thought to require a careful balance between cell recruitment and growth factor release kinetics. The objective of this study was to identify a growth factor combination that accelerates progenitor cell migration into self-assembling peptide hydrogels in the context of cartilage defect repair. A novel 3D gel-to-gel migration assay enabled quantification of the chemotactic impact of platelet-derived growth factor-BB (PDGF-BB), heparin-binding insulin-like growth factor-1 (HB-IGF-1), and transforming growth factor-ß1 (TGF-ß1) on progenitor cells derived from subchondral bovine trabecular bone (bone-marrow progenitor cells, BM-PCs) encapsulated in the peptide hydrogel [KLDL]3. Only the combination of PDGF-BB and TGF-ß1 stimulated significant migration of BM-PCs over a 4-day period, measured by confocal microscopy. Both PDGF-BB and TGF-ß1 were slowly released from the gel, as measured using their (125)I-labeled forms, and they remained significantly present in the gel at 4 days. In the context of augmenting microfracture surgery for cartilage repair, our strategy of delivering chemotactic and proanabolic growth factors in KLD may provide the necessary local stimulus to help increase defect cellularity, providing more cells to generate repair tissue.

Effects of Dexamethasone Concentration and Timing of Exposure on Chondrogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells.

OBJECTIVE: Dexamethasone is known to support mesenchymal stem cell (MSC) chondrogenesis, although the effects of dose and timing of exposure are not well understood. The objective of this study was to investigate these variables using a laboratory model of MSC chondrogenesis. DESIGN: Equine MSCs were encapsulated in agarose and cultured in chondrogenic medium with 1 or 100 nM dexamethasone, or without dexamethasone, for 15 days. Samples were analyzed for extracellular matrix (ECM) accumulation, prostaglandin E2 and alkaline phosphatase secretion, and gene expression of selected collagens and catabolic enzymes. Timing of exposure was evaluated by ECM accumulation after dexamethasone was withdrawn over the first 6 days, or withheld for up to 3 or 6 days of culture. RESULTS: ECM accumulation was not significantly different between 1 and 100 nM dexamethasone, but was suppressed ~40% in dexamethasone-free cultures. Prostaglandin E2 secretion, and expression of catabolic enzymes, including matrix metalloproteinase 13, and type X collagen was generally lowest in 100 nM dexamethasone and not significantly different between 1 nM and dexamethasone-free cultures. Dexamethasone could be withheld for at least 2 days without affecting ECM accumulation, while withdrawal studies suggested that dexamethasone supports ECM accumulation beyond day 6. CONCLUSION: One nanomolar dexamethasone supported robust cartilage-like ECM accumulation despite not having an effect on markers of inflammation, although higher concentrations of dexamethasone may be necessary to suppress undesirable hypertrophic differentiation. While early exposure to dexamethasone was not critical, sustained exposure of at least a week appears to be necessary to maximize ECM accumulation.

Addition of Mesenchymal Stem Cells to Autologous Platelet-Enhanced Fibrin Scaffolds in Chondral Defects: Does It Enhance Repair?

BACKGROUND: The chondrogenic potential of culture-expanded bone-marrow-derived mesenchymal stem cells (BMDMSCs) is well described. Numerous studies have also shown enhanced repair when BMDMSCs, scaffolds, and growth factors are placed into chondral defects. Platelets provide a rich milieu of growth factors and, along with fibrin, are readily available for clinical use. The objective of this study was to determine if the addition of BMDMSCs to an autologous platelet-enriched fibrin (APEF) scaffold enhances chondral repair compared with APEF alone. METHODS: A 15-mm-diameter full-thickness chondral defect was created on the lateral trochlear ridge of both stifle joints of twelve adult horses. In each animal, one defect was randomly assigned to receive APEF+BMDMSCs and the contralateral defect received APEF alone. Repair tissues were evaluated one year later with arthroscopy, histological examination, magnetic resonance imaging (MRI), micro-computed tomography (micro-CT), and biomechanical testing. RESULTS: The arthroscopic findings, MRI T2 map, histological scores, structural stiffness, and material stiffness were similar (p > 0.05) between the APEF and APEF+BMDMSC-treated repairs at one year. Ectopic bone was observed within the repair tissue in four of twelve APEF+BMDMSC-treated defects. Defects repaired with APEF alone had less trabecular bone edema (as seen on MRI) compared with defects repaired with APEF+BMDMSCs. Micro-CT analysis showed thinner repair tissue in defects repaired with APEF+BMDMSCs than in those treated with APEF alone (p < 0.05). CONCLUSIONS: APEF alone resulted in thicker repair tissue than was seen with APEF+BMDMSCs. The addition of BMDMSCs to APEF did not enhance cartilage repair and stimulated bone formation in some cartilage defects. CLINICAL RELEVANCE: APEF supported repair of critical-size full-thickness chondral defects in horses, which was not improved by the addition of BMDMSCs. This work supports further investigation to determine whether APEF enhances cartilage repair in humans.

Clinical outcome after intra-articular administration of bone marrow derived mesenchymal stem cells in 33 horses with stifle injury.

OBJECTIVE: To report outcome of horses with femorotibial lesions (meniscal, cartilage or ligamentous) treated with surgery and intra-articular administration of autologous bone marrow derived mesenchymal stem cells (BMSCs). STUDY DESIGN: Prospective case series. ANIMALS: Horses (n = 33). METHODS: Inclusion criteria included horses that had lameness localized to the stifle by diagnostic anesthesia, exploratory stifle arthroscopy and subsequent intra-articular administration of autologous BMSCs. Case details and follow-up were gathered from medical records, owner, trainer or veterinarian. Outcome was defined as returned to previous level of work, returned to work, or failed to return to work. RESULTS: Follow-up (mean, 24 months) was obtained; 43% of horses returned to previous level of work, 33% returned to work, and 24% failed to return to work. In horses with meniscal damage (n = 24) a higher percentage in the current study (75%) returned to some level of work compared to those in previous reports (60-63%) that were treated with arthroscopy alone, which resulted in a statistically significant difference between studies (P = .038). Joint flare post injection was reported in 3 horses (9.0%); however, no long-term effects were noted. CONCLUSIONS: Intra-articular administration of BMSC postoperatively for stifle lesions appeared to be safe, with morbidity being similar to that of other biologic agents. Improvement in ability to return to work may be realized with BMSC treatment compared to surgery alone in horses with stifle injury.

Sustained delivery of bioactive TGF-ß1 from self-assembling peptide hydrogels induces chondrogenesis of encapsulated bone marrow stromal cells.

Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor ß1 (TGF-ß1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-ß1, fivefold higher retention was found in peptide. Excess unlabeled TGF-ß1 minimally displaced retained radiolabeled TGF-ß1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly threefold lower than agarose hydrogels, at 18% of loaded TGF-ß1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-ß1 was 32-44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-ß1 degradation and release of small labeled species. TGF-ß1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.

Effects of equine bone marrow aspirate volume on isolation, proliferation, and differentiation potential of mesenchymal stem cells.

OBJECTIVE: To compare the mesenchymal stem cell (MSC) yield and chondrogenic and osteogenic differentiation from 5- and 50-mL bone marrow aspirates from horses. ANIMALS: Six 2- to 5-year-old mixed-breed horses. Procedures-2 sequential 5-mL aspirates were drawn from 1 ilium or sternebra. A single 50-mL aspirate was drawn from the contralateral ilium, and 2 sequential 50-mL aspirates were drawn from a second sternebra. The MSC yield was determined through the culture expansion process. Chondrogenesis and osteogenesis were evaluated by means of conventional laboratory methods. RESULTS: The second of the 2 sequential 50-mL sternal aspirates yielded few to no MSCs. Independent of location, the highest density of MSCs was in the first of the 2 sequential 5-mL fractions, although with subsequent culture expansion, the overall yield was not significantly different between the first 5-mL and first 50-mL fractions. Independent of location, chondrogenesis and osteogenesis were not significantly different among fractions. Independent of fraction, the overall cell yield and chondrogenesis from the ilium were significantly higher than that from the sternum. CONCLUSIONS AND CLINICAL RELEVANCE: This study failed to detect an additional benefit of 50-mL aspirates over 5-mL aspirates for culture-expanding MSCs for equine clinical applications. Chondrogenesis was highest for MSCs from ilial aspirates, although it is not known whether chondrogenesis is indicative of activation of other proposed pathways by which MSCs heal tissues.

Synoviocytes protect cartilage from the effects of injury in vitro.

BACKGROUND: It is well documented that osteoarthritis (OA) can develop following traumatic joint injury and is the leading cause of lameness and subsequent wastage of equine athletes. Although much research of injury induced OA has focused on cartilage, OA is a disease that affects the whole joint organ. METHODS: In this study, we investigated the impact of synovial cells on the progression of an OA phenotype in injured articular cartilage. Injured and control cartilage were cultured in the presence of synoviocytes extracted from normal equine synovium. Synoviocytes and cartilage were evaluated for catabolic and anabolic gene expression. The cartilage was also evaluated histologically for loss of extracellular matrix molecules, chondrocyte cell death and chondrocyte cluster formation. RESULTS: The results indicate synoviocytes exert both positive and negative effects on injured cartilage, but ultimately protect injured cartilage from progressing toward an OA phenotype. Synoviocytes cultured in the presence of injured cartilage had significantly reduced expression of aggrecanase 1 and 2 (ADAMTS4 and 5), but also had increased expression of matrix metalloproteinase (MMP) -1 and reduced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1). Injured cartilage cultured with synoviocytes had increased expression of both collagen type 2 and aggrecanase 2. Histologic examination of cartilage indicated that there was a protective effect of synoviocytes on injured cartilage by reducing the incidence of both focal cell loss and chondrocyte cluster formation, two major hallmarks of OA. CONCLUSIONS: These results support the importance of evaluating more than one synovial joint tissue when investigating injury induced OA.

Development of an in vitro model of injury-induced osteoarthritis in cartilage explants from adult horses through application of single-impact compressive overload.

OBJECTIVE: To develop an in vitro model of cartilage injury in full-thickness equine cartilage specimens that can be used to simulate in vivo disease and evaluate treatment efficacy. SAMPLE: 15 full-thickness cartilage explants from the trochlear ridges of the distal aspect of the femur from each of 6 adult horses that had died from reasons unrelated to the musculoskeletal system. PROCEDURES: To simulate injury, cartilage explants were subjected to single-impact uniaxial compression to 50%, 60%, 70%, or 80% strain at a rate of 100% strain/s. Other explants were left uninjured (control specimens). All specimens underwent a culture process for 28 days and were subsequently evaluated histologically for characteristics of injury and early stages of osteoarthritis, including articular surface damage, chondrocyte cell death, focal cell loss, chondrocyte cluster formation, and loss of the extracellular matrix molecules aggrecan and types I and II collagen. RESULTS: Compression to all degrees of strain induced some amount of pathological change typical of clinical osteoarthritis in horses; however, only compression to 60% strain induced significant changes morphologically and biochemically in the extracellular matrix. CONCLUSIONS AND CLINICAL RELEVANCE: The threshold strain necessary to model injury in full-thickness cartilage specimens from the trochlear ridges of the distal femur of adult horses was 60% strain at a rate of 100% strain/s. This in vitro model should facilitate study of pathophysiologic changes and therapeutic interventions for osteoarthritis.