Periostin regulation and cartilage degradation early after anterior cruciate ligament reconstruction.

OBJECTIVE AND DESIGN: The purpose of this study was to explore pathological processes during the first 4 weeks after anterior cruciate ligament reconstruction (ACLR). SUBJECTS: Sixteen ACL-injured patients (8 females/8 males, mean age = 19.1, mean BMI = 28.6). METHODS: Arthrocentesis was performed 1 and 4 weeks after ACLR. Proteins in the synovial fluid were identified using nanoLC-ESI-MS/MS. Differentially up- or down-regulated proteins were identified and quantified, and a pathway analysis was performed. All identified proteins were mapped into a protein-protein interaction (PPI) network, and networks of PPIs with a combined score > 0.9 were then visualized. RESULTS: Seven pathways were upregulated after ACLR: PI3K-AKT signaling pathway, extracellular matrix (ECM)-receptor interaction, focal adhesion, protein digestion and absorption, ameobiasis, and platelet activation. Network analyses identified 8 proteins that were differentially upregulated with strong PPI interactions (periostin and 7 collagen-related proteins). Increases in periostin moderately correlated with increases in a synovial fluid biomarker of type II cartilage degradation (ρ = 0.51, p = 0.06). CONCLUSION: Pro-inflammatory pathways and periostin were upregulated after ACLR. Periostin demonstrated strong network connections with markers of collagen breakdown, and future work is needed to determine whether periostin may offer a biomarker of early cartilage degradation after ACLR and/or play an active role in early post-traumatic osteoarthritis (PTOA) progression.

Equine Bone Marrow Aspirate Concentrate.

Bone marrow concentrate is generated by centrifugation of bone marrow aspirate. It contains mesenchymal stromal cells, anabolic chemokines/cytokines, and supraphysiological concentrations of interleukin-1 receptor antagonist protein (IL-1RA). It is an effective treatment for osteoarthritis or desmitis, or as an adjunct in surgery to enhance bone or cartilage repair.

Clinically Significant Outcomes Following the Treatment of Focal Cartilage Defects of the Knee With Microfracture Augmentation Using Cartilage Allograft Extracellular Matrix: A Multicenter Prospective Study.

PURPOSE: To determine the short-term outcomes following microfracture augmented with cartilage allograft extracellular matrix for the treatment of symptomatic focal cartilage defects of the adult knee. METHODS: Forty-eight patients enrolled by 8 surgeons from 8 separate institutions were included in this study. Patients underwent microfracture augmented by cartilage allograft extracellular matrix (BioCartilage; Arthrex, Naples, FL) and were followed at designated time points (3, 6, 12, and 24 months) to assess patient-reported outcomes (PROs), clinically significant outcomes (CSOs), and failure and complication rates. Magnetic resonance imaging (MRI) was offered at 2 years postoperatively regardless of symptomatology, and Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) 2.0 score was documented. RESULTS: PRO compliance was 81.3% at 6 months, 72.9% at 12 months, and 47.9% at 2 years. All joint-specific and function-related PROs significantly improved compared to baseline at 3, 6, 12, 18, and 24 months of follow-up (P < .01), apart from Marx activity scale, which demonstrated a significant decline in postoperative scores at 2 years (P = .034). The percentage of patients achieving CSOs (as defined for microfracture) at 2 years was 90% for minimal clinically important difference and 85% for patient acceptable symptomatic state. Patient factors including age, sex, body mass index, symptoms duration, smoking, presence of a meniscal tear, lesion size, and location were not associated with CSO achievement at 2 years. One patient (2.1%) failed treatment 9.5 months postoperatively due to graft delamination and required a reoperation consisting of arthroscopic debridement. One complication (2.1%) consisting of complaints of clicking, grinding, and crepitus 15 months following the index procedure was reported. Two-year postoperative MRI demonstrated a mean 40.5 ± 22.9 MOCART 2.0 score. CONCLUSIONS: In this preliminary study, we found cartilage allograft extracellular matrix to be associated with improvement in functional outcomes, high rates of CSO achievement, and low failure and complication rates at 2-year follow-up. LEVEL OF EVIDENCE: Level III, prospective multicenter cohort study.

Return to racing after surgical management of third carpal bone slab fractures in thoroughbred and standardbred racehorses.

OBJECTIVE: To determine the prognosis for racing of horses surgically treated for slab fractures of the third carpal bone (C3). STUDY DESIGN: Retrospective case study. ANIMALS: Horses (n = 125) surgically treated for C3 slab fractures. METHODS: Medical records of horses surgically treated for dorsal or sagittal C3 fractures were reviewed for age, sex, breed, limb, fracture type, degree of cartilage damage, and surgical treatment. Radiographs were evaluated to determine fracture depth, width, and displacement. Osteophytes, C3 lysis, and fragmentation were scored. Racing performance was obtained from online databases. Univariable and multivariable analyses were used to determine associations between independent variables and outcomes. RESULTS: Fifty-four (43%) horses raced postoperatively. Among thoroughbreds, 35% (30/86) with dorsal fractures and 63% (17/27) with sagittal fractures raced postoperatively. Among standardbreds, 77% (10/13) with dorsal fractures and 0% (0/2) with sagittal fractures raced postoperatively. Fracture displacement, C3 lysis, and cartilage damage affected the likelihood of racing postoperatively. Placement of 3.5-mm screws vs 4.5-mm screws and the placement of fewer screws were associated with improved likelihood of racing. CONCLUSION: The prognosis for postoperative racing of thoroughbreds with dorsal C3 fractures was less favorable than that previously reported. Concurrent joint pathology, such as cartilage damage at time of surgery, affected the ability of the horse to race postoperatively. CLINICAL SIGNIFICANCE: Although internal fixation of C3 slab fractures is required to restore joint congruity, return to racing should be expected in only 42% of thoroughbreds and 67% of standardbreds.

Bone marrow concentrate and platelet-rich plasma differ in cell distribution and interleukin 1 receptor antagonist protein concentration.

PURPOSE: Bone marrow concentrate (BMC) and platelet-rich plasma (PRP) are used extensively in regenerative medicine. The aim of this study was to determine differences in the cellular composition and cytokine concentrations of BMC and PRP and to compare two commercial BMC systems in the same patient cohort. METHODS: Patients (29) undergoing orthopaedic surgery were enrolled. Bone marrow aspirate (BMA) was processed to generate BMC from two commercial systems (BMC-A and BMC-B). Blood was obtained to make PRP utilizing the same system as BMC-A. Bone marrow-derived samples were cultured to measure colony-forming units, and flow cytometry was performed to assess mesenchymal stem cell (MSC) markers. Cellular concentrations were assessed for all samples. Catabolic cytokines and growth factors important for cartilage repair were measured using multiplex ELISA. RESULTS: Colony-forming units were increased in both BMCs compared to BMA (p < 0.0001). Surface markers were consistent with MSCs. Platelet counts were not significantly different between BMC-A and PRP, but there were differences in leucocyte concentrations. TGF-ß1 and PDGF were not different between BMC-A and PRP. IL-1ra concentrations were greater (p = 0.0018) in BMC-A samples (13,432 pg/mL) than in PRP (588 pg/mL). The IL-1ra/IL-1ß ratio in all BMC samples was above the value reported to inhibit IL-1ß. CONCLUSIONS: The bioactive factors examined in this study have differing clinical effects on musculoskeletal tissue. Differences in the cellular and cytokine composition between PRP and BMC and between BMC systems should be taken into consideration by the clinician when choosing a biologic for therapeutic application. LEVEL OF EVIDENCE: Clinical, Level II.

Effect of needle diameter on the viability of equine bone marrow derived mesenchymal stem cells.

OBJECTIVES: Mesenchymal stem cells (MSCs) are frequently delivered via needle injection for treatment of musculoskeletal injuries. The purpose of this study was to evaluate the effect of needle diameter on the viability of MSCs. METHODS: Equine bone marrow-derived MSCs from 5 horses were suspended in PBS, and held at room temperature for 7 hours to mimic shipping conditions. Two replicate samples for each needle size (20, 22, 23, or 25-gauge [ga]) were aspirated into a 3 mL syringe and re-injected into the holding vial 3 times, to reproduce the resuspension of cells prior to injection in clinical cases. Cells were stained with fluorescein diacetate and propidium iodide to measure viability. Flow cytometry (FC) was performed to compare cell debris and intact cells between groups. RESULTS: MSC viability was higher when cells were passed through a 20-ga rather than a 25-ga needle. Cell suspensions passed through a 20-ga needle contained a larger percentage of intact cells, compared to 25-ga samples. The percentage of debris present in cell suspensions tended to increase with decreasing needle diameter. Neither horse nor passage had a significant effect on viability. CONCLUSIONS: Cell damage is more likely when MSCs are passed through 25-ga rather than 20-ga needles. CLINICAL RELEVANCE: Use of needles larger than 25-ga is recommended to maintain the viability of MSCs injected in horses.

Editorial Commentary: Precise Nomenclature Is Critical for Evaluating Progress in the Field of Lipoaspirate-Derived Stem Cell Therapy.

In a Level III study, the addition of adipose-based cell therapy significantly improved the clinical outcome and arthroscopic appearance of articular cartilage in patients with osteoarthritic varus ankles that were treated with the combinational operative techniques of lateral sliding calcaneal osteotomy and microfracture. Use of adipose-based therapies from lipoaspirate is increasing, with many different types of processing available, resulting in very different biological products. The mesenchymal stem cell therapy terminology used in the article could be more precise, and the contribution of adipose cells to the outcome is confounded by the surgical procedures performed. On the positive side, the article is a good start to encourage the orthopaedic community to provide the evidence both in clinical outcome and in determining what is in the mixture of cells administered to the patient, so that the efficacy of adipose-based cell therapy can be transparent and critically evaluated.

The Effect of Different Bone Marrow Stimulation Techniques on Human Talar Subchondral Bone: A Micro-Computed Tomography Evaluation.

PURPOSE: To evaluate morphological alterations, microarchitectural disturbances, and the extent of bone marrow access to the subchondral bone marrow compartment using micro-computed tomography analysis in different bone marrow stimulation (BMS) techniques. METHODS: Nine zones in a 3 × 3 grid pattern were assigned to 5 cadaveric talar dome articular surfaces. A 1.00-mm microfracture awl (s.MFX), a 2.00-mm standard microfracture awl (l.MFX), or a 1.25-mm Kirschner wire (K-wire) drill hole was used to penetrate the subchondral bone in each grid zone. Subchondral bone holes and adjacent tissue areas were assessed by micro-computed tomography to analyze adjacent bone area destruction and communicating channels to the bone marrow. Grades 1 to 3 were assigned, where 1 = minimal compression/sclerosis; 2 = moderate compression/sclerosis; 3 = severe compression/sclerosis. Bone volume/total tissue volume, bone surface area/bone volume, trabecular thickness, and trabecular number were calculated in the region of interest. RESULTS: Visual assessment revealed that the s.MFX had significantly more grade 1 holes (P < .001) and that the l.MFX had significantly more poor/grade 3 holes (P = .002). Bone marrow channel assessment showed a statistically significant increase in the number of channels in the s.MFX when compared with both K-wire and l.MFX holes (P < .001). Bone volume fraction for the s.MFX was significantly less than that of the l.MFX (P = .029). CONCLUSIONS: BMS techniques using instruments with larger diameters resulted in increased trabecular compaction and sclerosis in areas adjacent to the defect. K-wire and l.MFX techniques resulted in less open communicating bone marrow channels, denoting a reduction in bone marrow access. The results of this study indicate that BMS using larger diameter devices results in greater microarchitecture disturbances. CLINICAL RELEVANCE: The current study suggests that the choice of a BMS technique should be carefully considered as the results indicate that smaller diameter hole sizes may diminish the amount of microarchitectural disturbances in the subchondral bone.

Platelet-rich plasma in the pathologic processes of cartilage: review of basic science evidence.

PURPOSE: The purpose of this study was to systematically review the basic science evidence for the use of platelet-rich plasma (PRP) in the treatment of pathologic processes of cartilage, both as an adjunct to cartilage repair and as a conservative management strategy for osteoarthritis, with the intent of determining the effect of PRP and whether a proof of concept for its use has been established to facilitate further investigation at a clinical level. METHODS: Using the terms "platelet-rich plasma OR PRP OR autologous conditioned plasma OR ACP AND cartilage OR chondrocytes OR chondrogenesis OR osteoarthritis OR arthritis" we searched EMBASE and PubMed/Medline in April 2012. Two authors performed the search, 3 authors independently assessed the studies for inclusion, and 2 authors extracted the data. Extracted data included cytologic analysis of PRP, study design, and results. RESULTS: Twenty-one studies (12 in vitro, 8 in vivo, one in vitro and in vivo) met the inclusion criteria. The effects of PRP in these studies included increasing chondrocyte and mesenchymal stem cell proliferation, proteoglycan deposition, and type II collagen deposition. PRP was also found to increase the cell viability of chondrocytes and the migration and chondrogenic differentiation of mesenchymal stem cells (MSCs) and to inhibit the effect of catabolic cytokines. In vivo, PRP was used as an adjunct to concomitant surgical management, including microfracture surgery and implant, scaffold, and graft insertion. Not all studies concluded that PRP has a positive effect on cartilage repair. CONCLUSIONS: The current basic science evidence suggests that PRP has several potential effects on cartilage repair and osteoarthritis, and a proof of concept has been established. Well-designed randomized controlled trials (RCTs) are needed to extrapolate this evidence to the clinical setting.

Interleukin-6 neutralization and regulatory T cells are additive in chondroprotection from IL-1ß-induced inflammation.

Anti-inflammatory Regulatory T cells (Tregs) are enriched in the joints of patients with osteoarthritis (OA) compared to healthy joints. Tregs maintain homeostasis through secretion of anti-inflammatory cytokines and cell-to-cell interactions including immune checkpoint signaling. Interleukin-6 (IL-6) is a pleiotropic cytokine secreted by inflamed synoviocytes and chondrocytes that can inhibit or alter Treg function. This study tested the hypothesis that neutralization of IL-6 would enable Treg anti-inflammatory function to resolve inflammation and catabolism elicited by IL-1ß in an equine chondrocyte/synoviocyte/Treg tri-culture OA model. Synoviocyte/chondrocyte co-cultures were stimulated with IL-1ß, and treated with αIL-6 neutralizing antibody. Activated Tregs secreting IL-10 were added in direct contact with synoviocytes to create a tri-culture. Neutralization of IL-6 partially restored Treg anti-inflammatory functions and, in combination, reduced IL-1ß-stimulated synoviocyte MMP13 expression to control levels and restored Acan expression in chondrocytes. IL-6 neutralization alone decreased Il6 expression in chondrocytes and synoviocytes, mitigating IL-6 positive feedback loop. Although Tregs were the primary producers of anti-inflammatory IL-10 and IL-4, they also produced pro-inflammatory IL-17A, as detected by ELIA, which may have been responsible for incomplete rescue of synoviocyte/chondrocyte homeostasis following IL-1ß stimulation. Treg secretion of IL-10, IL-4, and IL-17A was not altered by tri-culture conditions or presence of αIL-6, therefore, it was unlikely that Treg phenotype instability occurred. The significant effect of chondrocyte/synoviocyte donor, but not Treg donor, on gene expression and IL-6 concentration in conditioned media, indicated that personalized therapy considering the patient's OA status might be needed for successful implementation of immunotherapy in the context of OA.

T Helper 17-Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis.

BACKGROUND: Infiltration of cluster of differentiation (CD) 3+ (CD3+) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets. HYPOTHESIS: An imbalance of the ratio of regulatory T cells and T helper 17 cells would be associated with disease progression in posttraumatic osteoarthritis, suggesting opportunities for immunomodulatory therapy. STUDY DESIGN: Descriptive laboratory study. METHODS: Synovial fluid was aspirated from the joints of equine clinical patients undergoing arthroscopic surgery for posttraumatic osteoarthritis resulting from intra-articular fragmentation. Joints were classified as having mild or moderate posttraumatic osteoarthritis. Synovial fluid was also obtained from nonoperated horses with normal cartilage. Peripheral blood was obtained from horses with normal cartilage and those with mild and moderate posttraumatic osteoarthritis. Synovial fluid and peripheral blood cells were analyzed by flow cytometry, and native synovial fluid was analyzed by enzyme-linked immunosorbent assay. RESULTS: CD3+ T cells represented 81% of lymphocytes in synovial fluid, which increased in animals with moderate posttraumatic osteoarthritis to 88.3% (P = .02). CD14+ macrophages were doubled in those with moderate posttraumatic osteoarthritis compared with mild posttraumatic osteoarthritis and controls (P < .001). Less than 5% of CD3+ T cells found within the joint were forkhead box P3 protein+ (Foxp3+) regulatory T cells, but a 4- to 8-times higher percentage of nonoperated and mild posttraumatic osteoarthritis joint regulatory T cells secreted interleukin (IL)-10 than peripheral blood Tregs (P < .005). T regulatory-1 cells that secreted IL-10 but did not express Foxp3 accounted for approximately 5% of CD3+ T cells in all joints. T helper 17 cells and Th17-like regulatory T cells were increased in those with moderate posttraumatic osteoarthritis (P < .0001) compared with mild and nonoperated patients. IL-10, IL-17A, IL-6, chemokine (C-C motif) ligand (CCL) 2 (CCL2), and CCL5 concentrations detected by enzyme-linked immunosorbent assay in synovial fluid were not different between groups. CONCLUSIONS: An imbalance of the ratio of regulatory T cells and T helper 17 cells and an increase in T helper 17 cell-like regulatory T cells in synovial fluid from joints with more severe disease provide novel insights into immunological mechanisms that are associated with posttraumatic osteoarthritis progression and pathogenesis. CLINICAL RELEVANCE: Early and targeted use of immunotherapeutics in the mitigation of posttraumatic osteoarthritis may improve patient clinical outcomes.

Novel Osteochondral Biotemplate Improves Long-term Cartilage Repair Compared With Microfracture in an Ovine Model.

BACKGROUND: Current cartilage repair therapies do not re-create the complex mechanical interface between cartilage and bone, which is critical for long-term repair durability. New biomaterial designs that include hard tissue-soft tissue interface structures offer promise to improve clinical outcomes. PURPOSE/HYPOTHESIS: The purpose of this study was to evaluate the efficacy and safety of a naturally derived osteochondral biotemplate with a novel contiguous hard tissue-soft tissue interface in an ovine model as a regenerative solution for articular cartilage defects. It was hypothesized that the osteochondral biotemplate would produce structurally superior repair tissue compared with microfracture over a 13-month period. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral biotemplates were manufactured from porcine cancellous bone. Skeletally mature sheep (N = 30) were randomly allocated to 3 groups: early healing stage (euthanasia at 4 months), 6-month treatment, and 13-month treatment. In the early healing stage group, an 8 mm-diameter by 5 mm-deep osteochondral defect was created on the medial femoral condyle and treated at the time of iatrogenic injury with an osteochondral biotemplate. The contralateral limb received the same treatment 2 months later. In the 6- and 13-month treatment groups, 1 limb received the same osteochondral procedure as the early healing stage group. In the contralateral limb, an 8 mm-diameter, full-thickness cartilage defect (1-2 mm deep) was created and treated with microfracture. Cartilage repair and integration were quantitatively and qualitatively assessed with gross inspection, histological evaluation, and magnetic resonance imaging (MRI). Wilcoxon signed-rank and McNemar tests were used to compare the treatments. RESULTS: At 6 and 13 months after treatment, the biotemplate was not present histologically. At 13 months, the biotemplate treatment demonstrated statistically higher histological scores than microfracture for integration with surrounding cartilage (biotemplate: 74 ± 31; microfracture: 28 ± 39; P = .03), type 2 collagen (biotemplate: 72 ± 33; microfracture: 40 ± 38; P = .02), total cartilage (biotemplate: 71 ± 9; microfracture: 59 ± 9; P = .01), and total integration (biotemplate: 85 ± 15; microfracture: 66 ± 20; P = .04). The osteochondral biotemplate treatment produced a notable transient nonneutrophilic inflammatory response that appeared to approach resolution at 13 months. MRI results were not statistically different between the 2 treatments. CONCLUSION: Even with the inflammatory response, after 13 months, the osteochondral biotemplate outperformed microfracture in cartilage regeneration and demonstrated superiority in integration between the repair tissue and host tissue as well as integration between the newly formed cartilage and the underlying bone. CLINICAL RELEVANCE: This work has demonstrated the clinical potential of a novel biomaterial template to regenerate the complex mechanical interface between cartilage and the subchondral bone.

Composition and Bioactivity of a Placental Tissue Particulate (PTP-001) Indicate Greater Potential than Platelet-Rich Plasma for the Treatment of Osteoarthritis.

OBJECTIVE: This study was conducted to compare therapeutically relevant properties of platelet-rich plasma (PRP), a commonly used autologous intra-articular treatment for osteoarthritis (OA), with those of a novel placental tissue particulate, PTP-001, which is in development as a regulated biologic treatment for knee OA. DESIGN: Quantitative immunoassays were performed to determine the content of key growth/regulatory biofactors in PTP-001, and in leukocyte-rich (LR)-PRP or leukocyte-poor (LP)-PRP. An anti-inflammatory bioassay was used to evaluate the effects of each treatment on pro-inflammatory cytokine (tumor necrosis factor (TNF)-α) production in a macrophage cell culture system. Gene expression experiments were conducted using a co-culture system of human synoviocytes (pre-stimulated with interleukin (IL)-1ß) and articular chondrocytes, with quantitative polymerase chain reaction analyses of the separate cellular compartments. RESULTS: The concentrations of several biofactors (e.g., basic fibroblast growth factor, tissue inhibitor of metalloproteases-3, interleukin-1 receptor antagonist) representative of diverse disease-relevant mechanisms of action were significantly higher for PTP-001 relative to LR-PRP or LP-PRP. PTP-001 and PRP preparations were able to reduce TNF-α production in macrophage cell cultures; however, greater variability was observed for PRP in comparison with PTP-001. In the chondrocyte/synoviocyte co-culture experiments, PTP-001 and LR-PRP (but not LP-PRP) significantly reduced chondrocyte MMP13 expression in cultures containing IL-1-pretreated synoviocytes. In addition, ADAMTS5 expression was reduced in the chondrocyte compartment following treatment with PTP-001 relative to PRP. CONCLUSION: These findings support evidence of a potent, multifactorial mechanism of action for a consistently manufactured biologic (PTP-001), which may be of greater therapeutic benefit in comparison with more heterogeneous preparations of PRP which may be generated at the time of treatment.

Complement system dysregulation in synovial fluid from patients with persistent inflammation following anterior cruciate ligament reconstruction surgery.

Introduction: Patients with anterior cruciate ligament injury are at high risk of posttraumatic osteoarthritis and their response to reconstructive surgery and rehabilitation vary. Proteins identified in the orchestration of the acute inflammatory response may be predictive of patient outcomes. Objective: An unbiased, bottom-up proteomics approach was used to discover novel targets for therapeutics in relation to dysregulation in the orchestration of inflammatory pathways implicated in persistent joint inflammation subsequent to joint trauma. Methods: Synovial fluid was aspirated from patients at 1 week and 4 weeks after anterior cruciate ligament reconstruction (ACLR) and interleukin 6 (IL-6) concentrations were quantified by enzyme-linked immunosorbent assay. Patients were segregated into IL-6low and IL-6high groups based on IL-6 concentrations in synovial fluid at 4-weeks postoperation and proteins in synovial fluid were analyzed using qualitative, bottom-up proteomics. Abundance ratios were calculated for IL-6high and IL-6low groups as 4 weeks postoperation:1 week postoperation. Results: A total of 291 proteins were detected in synovial fluid, 34 of which were significantly (P < .05) differentially regulated between groups. Proteins associated with the classical and alternative complement cascade pathways were increased in the IL-6high compared to IL-6low group. Insulin-like growth factor-binding protein 6 (IGFBP-6) was increased by nearly 60-fold in the IL-6low group. Conclusions: Patients segregated by IL-6 concentration in synovial fluid at 4 weeks post-ACLR demonstrated differential regulation of multiple pathways, providing opportunities to investigate novel targets, such as IGFBP-6, and to take advantage of therapeutics already approved for clinical use in other diseases that target inflammatory pathways, including the complement system.

Platelet-rich plasma: a milieu of bioactive factors.

Platelet concentrates such as platelet-rich plasma (PRP) have gained popularity in sports medicine and orthopaedics to promote accelerated physiologic healing and return to function. Each PRP product varies depending on patient factors and the system used to generate it. Blood from some patients may fail to make PRP, and most clinicians use PRP without performing cell counts on either the blood or the preparation to confirm that the solution is truly PRP. Components in this milieu have bioactive functions that affect musculoskeletal tissue regeneration and healing. Platelets are activated by collagen or other molecules and release growth factors from alpha granules. Additional substances are released from dense bodies and lysosomes. Soluble proteins also present in PRP function in hemostasis, whereas others serve as biomarkers of musculoskeletal injury. Electrolytes and soluble plasma hormones are required for cellular signaling and regulation. Leukocytes and erythrocytes are present in PRP and function in inflammation, immunity, and additional cellular signaling pathways. This article supports the emerging paradigm that more than just platelets are playing a role in clinical responses to PRP. Depending on the specific constituents of a PRP preparation, the clinical use can theoretically be matched to the pathology being treated in an effort to improve clinical efficacy.

Science and animal models of marrow stimulation for cartilage repair.

Microfracture of subchondral bone to enhance cartilage repair is a popular surgical technique used in human and animal patients. Clinical results with resolution or improvement in pain are promising and last on average for 2 to 3 years. Animal studies aimed at understanding microfracture indicate that the repair tissue continues to remodel toward chondrogenesis for at least a year, but longer term results are not available to gain insight into the mechanism of microfracture function or failure over time. Subchondral bone sclerosis and central lesional osteophyte formation following subchondral bone microfracture have been observed in animal models of microfracture, but studies do not provide any insight into the etiology of these pathologies. The continued maturation of microfracture repair tissue over time supports further investigation of microfracture or microfracture-augmented cartilage repair procedures with caution for the investigator and clinician to be observant for conditions that lead to subchondral bone sclerosis or central osteophyte formation, and what affect these boney reactions have on clinical outcome.

STRAINS: A big data method for classifying cellular response to stimuli at the tissue scale.

Cellular response to stimulation governs tissue scale processes ranging from growth and development to maintaining tissue health and initiating disease. To determine how cells coordinate their response to such stimuli, it is necessary to simultaneously track and measure the spatiotemporal distribution of their behaviors throughout the tissue. Here, we report on a novel SpatioTemporal Response Analysis IN Situ (STRAINS) tool that uses fluorescent micrographs, cell tracking, and machine learning to measure such behavioral distributions. STRAINS is broadly applicable to any tissue where fluorescence can be used to indicate changes in cell behavior. For illustration, we use STRAINS to simultaneously analyze the mechanotransduction response of 5000 chondrocytes-over 20 million data points-in cartilage during the 50 ms to 4 hours after the tissue was subjected to local mechanical injury, known to initiate osteoarthritis. We find that chondrocytes exhibit a range of mechanobiological responses indicating activation of distinct biochemical pathways with clear spatial patterns related to the induced local strains during impact. These results illustrate the power of this approach.

Synovium secretome as a disease-modifying treatment for equine osteoarthritis.

OBJECTIVE: To identify chondroprotective factors as potential disease-modifying osteoarthritis treatments using an unbiased, bottom-up proteomics approach. SAMPLES: Paired equine cartilage explants and synovial membrane were collected postmortem from 4 horses with no history of lameness and grossly normal joints at necropsy. PROCEDURES: Six groups were established: cartilage, synoviocytes, and cartilage + synoviocytes (coculture), all with or without interleukin (IL)-1ß. The catabolic effect of IL-1ß was verified by glycosaminoglycan (GAG) released from cartilage into media by 1,9-dimethyl-methylene blue assay and cartilage toluidine blue histochemistry. Conditioned media from cocultures with or with IL-1ß were submitted for bottom-up proteomic analysis. Synoviocyte gene expression was evaluated using reverse transcription-quantitative PCR (RT-qPCR) for proteins of interest identified in the proteomics scan. RESULTS: GAG content was retained in cartilage when in cocultures treated with IL-1ß. Fourteen proteins of interest were selected from the proteomic analysis. From these 14 proteins, metalloproteinase inhibitor 3 precursor (TIMP3), tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), insulin-like growth factor-binding protein 2 (IGFBP2), and alpha-2 macroglobulin (A2M) were selected for synoviocyte gene expression analysis by RT-qPCR. Gene expression of TIMP3 (P = .02) and TNFRSF11B (P = .04) were significantly increased in synoviocytes from cocultures treated with IL-1ß compared to controls. Contrary to expectations based on protein expression, IGFBP2 gene expression (P = .04) was significantly decreased in IL-1ß-stimulated coculture synoviocytes compared to control coculture synoviocytes. A2M gene expression in synoviocytes was not different between coculture groups. CLINICAL RELEVANCE: The secretome from synoviocytes could provide a milieu of bioactive factors to restore joint homeostasis in osteoarthritis.

Structural origins of cartilage shear mechanics.

Articular cartilage is a remarkable material able to sustain millions of loading cycles over decades of use outperforming any synthetic substitute. Crucially, how extracellular matrix constituents alter mechanical performance, particularly in shear, remains poorly understood. Here, we present experiments and theory in support of a rigidity percolation framework that quantitatively describes the structural origins of cartilage's shear properties and how they arise from the mechanical interdependence of the collagen and aggrecan networks making up its extracellular matrix. This framework explains that near the cartilage surface, where the collagen network is sparse and close to the rigidity threshold, slight changes in either collagen or aggrecan concentrations, common in early stages of cartilage disease, create a marked weakening in modulus that can lead to tissue collapse. More broadly, this framework provides a map for understanding how changes in composition throughout the tissue alter its shear properties and ultimate in vivo function.