Purpose

Transplantation of genetically modified bone marrow concentrates is an attractive strategy to conveniently activate the chondrogenic differentiation processes as a means to improve the intrinsic repair capacities of damaged articular cartilage. In this study, we examined the potential benefits of co-overexpression of the pleiotropic fibroblast growth factor-2 (FGF-2) and the cartilage-specific transcription factor sox9 using recombinant adeno-associated virus (rAAV) vectors upon the chondroreparative processes in human bone marrow aspirates.

Methods and Materials

Human bone marrow aspirates were obtained from the distal femurs of patients undergoing total knee arthroplasty. Aspirates were aliquoted in 96-well plates and immediately transduced with the rAAV vectors (rAAV-lacZ: 20 or 40 μl) or co-transduced (rAAV-hFGF-2 /rAAV-FLAG-hsox9: 10 or 20 μl each vector) with each aliquot. 60 μl chondrogenic medium was then added per aspirate. To assess FGF-2 secretion, 30 μl of culture supernatant were collected at the denoted time points 24 h after medium change and FGF-2 production was measured by ELISA. Transgene expression, and expression of type-II, -I, and -X collagen were assessed by immunohistochemical analyses. Hematoxylin eosin for cellularity, toluidine blue for matrix proteoglycans, and alizarin red for matrix mineralization were also measured. Each condition was performed in duplicate in three independent experiments.

Results

Successful FGF-2/sox9 combined gene transfer and overexpression via rAAV was achieved in chondrogenically induced human bone marrow aspirates for up to 21 days, the longest time point evaluated, leading to increased proliferation, matrix synthesis, and chondrogenic differentiation relative to control treatments (reporter lacZ treatment, absence of vector application) especially when co-applying the candidate vectors at the highest vector doses tested. Moreover, optimal co-administration of FGF-2with sox9 also advantageously reduced hypertrophic differentiation in the aspirates.

Conclusion

These findings report the possibility of directly modifying bone marrow aspirates by combined therapeutic gene transfer as a potent and convenient future approach to improve the repair of articular cartilage lesions.

Purpose

Chitosan oligomers at various degrees of deacetylation (DDA) can modulate macrophages in vitro to release anti-inflammatory factors [1] and could potentially polarize synovial macrophages to an anti-inflammatory phenotype. This study evaluated the effect of intra-articular (IA) injections of chitosan oligomers administered post-operatively after bone marrow stimulation (BMS) of a chronic cartilage defect. We tested the hypothesis that oligomers alter synovial fluid composition, synovial macrophage phenotype, and early cartilage tissue regeneration, compared to saline-only and Synvisc® as a gold standard.

Methods and Materials

Following ethics-approved protocols, 4x4 mm bilateral cartilage defects were created in the femoral trochlea of skeletally mature NZW rabbits (N=12), brought to chronicity over four weeks, then microdrilled (four 0.9 mm diameter holes, 2 mm deep). At 1-week post-BMS, rabbits received 3 weekly IA-injections: 80% or 99% DDA chitosan formulation, saline (-CNL) or Synvisc®. Synovial fluid collected prior to surgeries and at necropsy was analyzed for hyaluronic acid concentration and differential cell counts. Synovial and cartilage repair tissues were analyzed histologically and repairing defects imaged ex vivo by high-frequency ultrasound.

Results

After 3 weeks of post-operative IA-injections, hyaluronic acid concentration was above 2 mg/mL and elevated relative to baseline at 0.72 mg/mL. Synovial fluid monocyte counts increased for all conditions except for 80% DDA chitosan that elicited a high neutrophil count (Fig. 1). Synovitis was observed for all conditions, but macrophage infiltration (RAM-11+) and vascularity were lower following 99% DDA oligomers IA-injections. Saline-injected knees had the most SafO+ cartilage repair tissues, suggesting that all biomaterials slowed chondro-induction (Fig. 2). Ultrasound imaging showed that drill holes were still repairing (Fig. 2).

Conclusion

Post-BMS IA-injection of 99% DDA oligomers showed a similar outcome as Synvisc® in decelerating the repair rate but with a better potential for reducing synovitis induced by BMS.

Acknowledgements: Jun Sun, Quentin O'Kelly. Funding: CIHR, NSERC, GMU startup.

References: [1] Fong (2017).

Purpose

The purpose of this study is to evaluate the efficacy of human synovial mesenchymal stem cells (MSCs) for cartilage regeneration in surgical-induced osteoarthritis (OA) in Beagle dogs.

Methods and Materials

Sixty Beagle dogs underwent meniscal injury for OA models. Every intra-articular injection was performed at 4 weeks after knee injury. The dogs were divided into five groups (n = 12) according to the intra-articular injection materials: the sham group without injection (G1), the control group with phosphate-buffered saline (G2), the group with low dose MSCs (2.4 × 10⁶ cells; G3), the group with medium dose MSCs (4.8 × 10⁶ cells; G4), and the group with high dose MSCs (9.6 × 10⁶ cells; G5). For evaluation of cartilage regeneration, histopathologic examinations that assessed the severity of cartilage damage, staining with Hematoxylin-eosin and Safranin-O, and analysis of immunohistochemistry (collagen type I and II) were used to document changes at time points of 6 or 24 weeks after injections.

Results

Severity of cartilage damage was significantly decreased in G3, G4, and G5 compared with G1 as well as G2 at both time points, and further improvements were observed at 24 weeks after injury compared with 6weeks after injury. Similar results were observed in staining with Hematoxylin-eosin and Safranin-O. Collagen composition of extracellular matrix (ECM) was more favorable in the G3, G4 and G5 than G1 as well as G2, and there were significant differences between groups (G3, G4 and G5); in other words, as the number of MSCs increased, more favorable results of collagen composition were observed. Further improvements in collagen composition were observed at 24 weeks after injury compared with 6weeks after injury.

Conclusion

Taken together, this study suggests that intra-articular injection of human synovial MSCs have a beneficial effect on OA via the chondrocyte proliferation and stimulation of ECM synthesis.

Purpose

Although the meta-analysis of the efficacy of Platelet-rich plasma (PRP) therapy for osteoarthritis (OA) shows favorable result, few studies have been reported the influence of administration of antiplatelet agents on the PRP therapy. Therefore, the aim of this study was to evaluate the quality of PRP and the efficacy of PRP therapy in the elderly patients with knee OA taking antiplatelet drugs.

Methods and Materials

115 patients (aged ≥60 years, 28 males, 87 females) who received more than 3 intra-articular PRP injections at our facility were enrolled. The patients were divided into two groups: patients taking antiplatelet drugs (group AP, n=29); patients without antiplatelet drugs (group N, n=86). The platelet in whole blood and PRP (total 718 samples, AP: 134, N: 584) were counted. Knee radiographs were graded using Kellgren-Lawrence (KL) classification. Clinical outcome was evaluated using the Visual Analog Scale (VAS) and the Japan Knee Osteoarthritis Measure (JKOM) questionnaire before treatment and at 3 months after treatment. Student’s t-test was used to compare the two groups. P values < 0.05 were considered statistically significant.

Results

There was no significant difference between the two groups regarding the patient’s age (AP: 72.8±0.6 years, N: 74.2±1.6 years, p=0.29) and OA severity (AP: KL4 64.0%, KL3 32.0%, KL≤2 4.9%; N: KL4 62.5%, KL3 21.4%, KL≤2 16.1%, Chi-squared test p=0.24). The platelet counts in whole blood (AP: 195.9±5.1 [x10⁹/L], N: 207.6±3.0, p=0.08) and PRP (AP: 444.8±15.3, N: 467.6±8.0, p=0.21) were not significantly different between the two groups (Fig.1). Regarding the clinical outcome, the improvement rate of VAS (AP: 30.5±30.9%, N: 21.1±50.0%, p=0.72) and JKOM (AP: 17.0±49.4 %, N: 10.3±35.6%, p=0.13) were not significantly different between the two groups (Fig.2).

Conclusion

These findings demonstrate that antiplatelet therapy would not affect the quality of PRP and the efficacy of PRP therapy.

Purpose

Bone marrow aspirate (BMA) is a promising treatment for knee OA. BMA contains a small percentage of stem cells, and this number is affected by quality of tissue harvesting, depending on the use of multiple aspiration sites with the collection of small volumes. Marrow-Cellution allows collecting high quality BMA using one access site, reducing harvesting time and procedure-related risks. The purpose of this study is to evaluate the mesenchymal (MSC) and hemopoietic (HSC) stem cells content in Marrow-Cellution-obtained BMA and its efficacy in an in vitro model of cartilage inflammation. These results will be compared with clinical outcomes of donor patients, undergoing intra-articular BMA injection.

Methods and Materials

BMA was obtained from iliac crest and tibia of 9 patients using Marrow-Cellution. Cell count, viability and percentage of stem cells was determined. A model of cartilage inflammation was established using IL-1β to stimulate human chondrocytes. Proliferation and immunophenotype of BMA cultured cells were assessed.

Results

The expected concentration of nucleated cells in native bone marrow is 1.5-3.0x10⁷ cells/ml. BMA from iliac crest revealed a total count of 1.3x10⁷ cells/ml, while 4x10⁶ cells/ml were found in the matched BMA from tibia. The percentage of HSCs and MSCs in BMA from iliac crest resulted 1.2% and 0.009%, respectively, similar to native bone marrow (1.5% HSCs, 0.01% MSCs). Tibia samples showed 0.06% HSCs and 0.002% MSCs. BMA cells proliferate in culture and possess the MSCs immunophenotype. BMA reduced the expression of catabolic markers in IL-1β-treated chondrocytes.

Conclusion

Marrow-Cellution allows collecting high quality BMA from iliac crest, showing similar cell count and percentage of stem cells as native bone marrow. The model of cartilage inflammation provided insights in BMA mechanism of action. The comparison of these results with the clinical outcomes will provide evidences about the BMA quality standards required to obtain significant results in the orthopedic practice.

Purpose

Indian hedgehog (Ihh) is known to control hypertrophic differentiation process of chondrocytes, its role from the mesenchyme cells to the early stages of chondrogenesis is unclear. To test the role of Ihh in the mesenchyme cells to the earlier stages of chondrogenesis, we have generated Prx1-Cre;Ihh^fl/fl;Rosa26^-ZsGreen1 mice to precisely delete Ihh in the mesenchyme cells. we can also track and isolate the deleted Ihh mesenchyme with EGFP. Thus, we can determine the role of Ihh from the mesenchyme to the earlier stage of chondrogenesis and bone formation in vivo.

Methods and Materials

The Prx1-Cre;Ihh^fl/+;Rosa26^-ZsGreen1 male mice were interbred with female Ihh^fl/fl mice to obtain Prx1Cre;Ihh^fl/fl;Rosa26^-ZsGreen1 animals.Safranin-O staining was performed to assess glycosaminoglycan production and von Kossa staining was performed to evaluate the mineralization of bone. RNA in situ hybridization for Collagen X mRNA was performed using the RNAscope® 2.0 paraffin embedded (FFPE) Reagent Kit. The complete bone marrow cavity of the tibia was evaluated by micro-CT.

Results

In situ hybridization on paraffin sections prepared from tibia at P0, a strong Collagen X mRNA was found in the whole medullary cavities in the mutant tibia while collagen X mRNA was only presented in pre-hypertrophic zone in control. Hypertrophic chondrocytes are located at hypertrophic zone without ossification in control mice, while whole cartilage template is von Kossa positive staining in mutant mice (Fig 1). The GFP positive cells that were derived from deleted Ihh mesenchyme cells overlap with von Kassa and Osteocalcin positive staining area. Bone histomorphometric analyses indicated that deleted Ihh results in more and disorganized trabecular formation (Fig 2).

Conclusion

Our findings demonstrate that deleting Ihh in mesenchyme cells during early limb development promote chondrocyte hypertrophy and pathological matrix calcification

Purpose

Osteochondral autograft transplantation (OAT) is a commonly performed surgical procedure in those suffering articular cartilage defects; however, limited literature exists with respect to return to sport and work timelines. The purpose of this study was to utilize an institutional cohort with minimum follow-up of 5 years to examine return to sport and work timelines following OAT.

Methods and Materials

A prospectively maintained institutional database was reviewed for all 45 patients undergoing OAT between January 2005 and April 2014 with a minimum follow-up of 5 years. Patients were contacted via phone with regard to pre- and post-operative participation in sport and work. Variables collected include demographics, surgical history, occupation, duty status, satisfaction, and the frequency, intensity and time to return to sports. SANE was also collected at the time of phone follow-up.

Results

A total of 21 patients (33.69±11.27 years, 42.86% right-sided) were contacted at a mean follow-up time of 10.54±2.86 years. Average lesion size was 1.22±0.47 cm². Fifty percent of patients underwent concomitant procedures; 25% were realignment procedures. The average SANE score at follow-up was 72.11±20.30, with 66.67% of patients being “Satisfied” or “Very Satisfied”. Survivorship to eventual knee replacement was 90.48% at 10.54 years, and 42.9% required subsequent surgery, most commonly cartilage debridement. 76.19% played sports preoperatively, with 81.25% returning to sports successfully at 7.68±3.99 months. Recreational athletes were more likely than competitive athletes to return to their main sport (84.62% vs. 37.5%, p=0.026). Eighty-one percent maintained an occupation preoperatively, with 76.5% returning to work at an average 2.98±3.13 months. Worker’s compensation patients were significantly less likely to return to work successfully, irrespective of duty status (p=0.046).

Conclusion

Following OAT, competitive athletes are less likely to return to their primary sport when compared to recreational athletes. Worker’s compensation patients are also less likely to return to work than those suffering non-worker’s compensation injury.

Purpose

Autologous blood derived products gain interest in the field of orthopaedics due to their ability to stimulate cartilage repair. Platelet rich plasma (PRP) is the most commonly used blood derivative in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. Drawbacks of using PRP are high donor variability, discrepancies among preparation protocols and the presence of cells which can evoke inflammation when injected into the defect. One possibility is to isolate only the active components of blood derivatives to overcome this problem. Extracellular vesicles (EVs) are particles transporting bioactive molecules and represent novel mechanisms by which signals are communicated among different cell types. In the current study we focused on EVs isolated from PRP and hyperacute serum, the latter mimicing the natural clotting cascade, in order to determine whether these particles impact the biological mode of action of blood derived products.

Methods and Materials

EVs were isolated from citrate-anticoagulated PRP (CPRP) and hyperacute serum using differential ultracentrifugation and size exclusion chromatography. Particle concentration and size were determined by nanoparticle tracking analysis (NTA). EVs were visualized by cryo-electronmicrosopy. Expression of miRNAs within EVs and in their respective input materials was analysed by RT-qPCR.

Results

NTA and cryo- electronmicroscopy revealed higher particle concentrations and bigger sized EVs within CPRP compared to hyperacute serum. Differences were detected regarding EV-marker and miRNA expression between the blood derivatives. The correlation between miRNAs in EVs and input material was higher in CPRP compared to hyperacute serum meaning that in hyperacute serum miRNAs were identified which were higher expressed in EVs than in the corresponding input material.

Conclusion

EVs from autologous blood products represent a novel and cell free approach for cartilage repair strategies. We observed that the clotting cascade (plasma versus serum) has an influence on concentration, size and miRNA expression patterns of EVs.

Purpose

To evaluate the use of trypsin enzymatic pretreatment of the surrounding cartilage combined with a self-assembling KLD hydrogel scaffold functionalized with growth factors for microfracture augmentation in an equine model.

Methods and Materials

Bilateral, 15 mm diameter cartilage defects were created on the medial trochlear ridge of the femoropatelar joints in eight adult horses (A, B, C). One defect was randomly assigned to receive microfracture plus treatment, while the other received microfracture only. Treatment consists of enzymatic exposure of the surrounding cartilage to trypsin for 2 minutes, followed by inactivation with fetal bovine serum (D). Standard subchondral bone microfracture was performed (E) and the defect was filled with self-assembling KLD hydrogel pre-mixed with growth factors (platelet-derived growth factor BB and heparin-binding insulin-like growth factor 1)(F). After surgery, all horses were submitted to standardized controlled exercises on a high-speed treadmill. Clinical evaluation and radiographic exams were conducted at multiple time points during the study period. After 12 months, all animals were euthanized and MRI, arthroscopy, gross pathologic evaluation of the joint, histology, immunohistochemistry and biomechanical analysis were performed. Generalized linear mixed models with horse as random effect were utilized to assess outcome parameters. When p-value<0.05, pairwise comparisons were made using least square means.

Results

Treatment resulted in improved functional outcome parameters (lameness and flexion test), even though mildly increased joint effusion and subchondral bone sclerosis was noticed on imaging. Microscopically, treatment resulted in overall improved histology scores and proteoglycan content in the proximal aspect of the reparative tissue, which is subjected to higher load. Further, dynamic compressive force and shear stiffness were increased in the cartilage adjacent to the treated defects.

Conclusion

Trypsin enzymatic pretreatment combined with a functionalized self-assembling KLD hydrogel resulted in improved cartilage healing and better functional outcomes compared to microfracture alone.

Purpose

Cartilage defects compromise tissue function, and often propagate and progress to osteoarthritis. The purpose of this study was to establish a microenvironment at the damaged interface that recruits and directs cells, with the goal of forming a living fibrous barrier to restore biomechanical function and prevent matrix loss.

Methods and Materials

Methacrylated hyaluronic acid (MeHA) was modified to include aldehydes (adhesiveness to tissue), FITC peptides (visualization), and fibronectin-mimicking peptides (RGD; for cellular adhesion). Bovine cartilage plugs were either defected or defected and digested in order to mimic focal defects (ND) and degenerated defects (D), respectively, and subject to biomaterial application and cross-linking (0, 5, 15 min). Engineered tissues were seeded with bovine mesenchymal stem cells (MSCs) and adhesion was evaluated at 24hr. The fibrogenic properties of these attached cells were evaluated by the presence of alpha-smooth muscle actin (ASMA) fibers at 7d. Finally, matrix production by adherent cells was visualized by the incorporation of azidohomoalanine (AHA) in place of L-methionine into newly synthesized proteins.

Results

The biomaterial diffused into and formed an integrated tissue-gel composite with cartilage, allowing for MSC attachment to the damaged interface (Fig 1A). Focal adhesion staining (Fig 1B) showed that biomaterial application and cross-linking increased the number of adhesions and adhesion area per cell (Fig 1C). Biomaterial with cross-linking (15 minutes) promoted fibrogenesis of MSCs in both focally defected and degenerated tissues (Fig 2A), with a greater number of ASMA positive cells (Fig 2B). Finally, increased matrix production was observed by cells cultured on engineered cartilage surfaces (Fig 2C/D).

Conclusion

This study demonstrated the ability of a modified HA biomaterial to form an integrated environment at the cartilage defect interface, enhancing the attachment, response, and matrix production of MSCs. Future studies will evaluate the ability of a living barrier to form and restore cartilage biomechanics in vitro and in vivo.