Purpose

Allogeneic equine bone marrow-derived mesenchymal stem cells (BMDMSCs) offer multiple clinical advantages over autologous BMDMSCs. Prior to clinical use, the safety and effectiveness of allogeneic versus autologous BMDMSCs must be determined. The purpose of the study was to evaluate the normal equine joint and inflamed equine joint reaction to allogeneic and autologous BMDMSCs.

Methods and Materials

Eight horses were used in the study. Autologous and pooled-allogeneic BMDMSCs were culture expanded, cryopreserved and thawed prior to administration. Clinical parameters (lameness, joint circumference and joint effusion) and synovial fluid parameters including nucleated cell count, differential cell count, total protein, prostaglandin E₂ (PGE₂) and C-reactive protein (CRP) were measured prior to intra-articular administration and 6, 12, 24, 72, 168, and 336 hours post-injection. Ten million autologous and allogeneic BMDMSCs were administered into contralateral (non-inflamed) metacarpophalangeal joints. After a 3 week washout period, 75ng of recombinent equine interleukin-1β (rIL-1β) and 10 million autologous BMDMSCs were administered into one tibiotarsal joint. After an additional one week period, the contralateral tibiotarsal joint received allogeneic BMDMSC plus 75ng rIL-1β. Four additional horses received 75ng rIL-1β alone in a single tibiotarsal joint.

Results

No difference was detected in any clinical or cytological parameters between (non-inflamed) metacarpophalangeal joints administered autologous or allogeneic BMDMSCs. In addition, no difference was detected in subjective lameness, joint effusion, joint circumference, NCC, TP, differential cell count, CRP or PGE₂ of inflamed joints administered autologous versus allogeneic BMDMSCs. Neither autologous nor allogeneic BMDMSCs resulted in any improvement in clinical or cytological parameters when compared to rIL-1β alone.

Conclusion

The study found no clinical or cytological differences in the normal joint response to autologous or allogeneic BMDMSCs. In addition, the study revealed no difference in the inflamed equine joint response to autologous or allogeneic BMDMSCs. Further, autologous and allogeneic BMDMSCs were equally ineffective in reducing the inflammatory response resulting from rIL-1β administration.

Purpose

Osteoarthritis(OA) treatment using mesenchymal stromal cells(MSC) has been extensively used and still there is no consensus regarding the number of cells applications and the best transplantation route. Using destabilization of the medial meniscus(DMM) as a model of OA, the aims of this study was to track MSC after intra-articular(IA) and intravenous(IV) injections, analyzing cytokines kinetics and histological structure.

Methods and Materials

OA was induced in female Wistar Kyoto rats via the DMM surgery. Rat bone marrow MSC was transduced with Luciferase transgene. Animals were treated with MSC after 9 weeks of surgery, via IA-MSC (2x10⁶cells/kg) and IV-MSC (4x10⁶cells/kg) injection. In vivo assay was acquired using the imaging system IN-VIVO MSFX PRO and the luminescence kinetic curve. Serum cytokines concentrations were determined with multiplex assay. Images and cytokines were analyzed at time intervals between 2h to 1 week after the transplant. After 4 and 12 weeks post-MSC treatment, knee was assessed by cartilage histology by OARSI score. Statistical analyzes were performed by Student's t-Test, one/two-way ANOVA.

Results

Viable MSC was observed in the OA knee until 1 week after the IA-MSC injection, and only until 8h in IV-MSC injection. The cytokines showed a different fluctuation concentration depended on the method of administration and time. IV-MSC injection increases MIP-1alpha and IL10 after 2h, decreasing in 24h, while MCP1 decreases after 2h. The opposite was observed with IA-MSC injection which increases MCP1 at the same time point, in addition to increase IL18 after 24h. Both cytokines decreased after 1 week. The OARSI score was better in both cell treatments than placebo. However, the IA-MSC showed a trend to reduce cartilage degeneration over time.

Conclusion

There is a possible homing capacity of MSC to the OA injury joint when injected IV. Taken together the results suggest that there is a longer lasting effect of MSC when injected via IA.

Purpose

Catwalk gait analysis system developed by Noldus records the paw prints of the rats walking in the band, they can classify them automatically and create dozens of parameters.

The purpose of this study is to evaluate whether the knees are completely healthy and that they are created chemically osteoarthritis with monosodiumiodoacetate in their knees firstly and then treated with intraarticular pharmacological agents such as hyaluronic acid, corticosteroid and astaxanthin.

Methods and Materials

In other studies, chemical osteoarthritis can be formed on animal models with different chemicals. In our study, monosodiumiodoacetate was preferred for chemical osteoarthritis in our experiment. We waited three weeks for chemical osteoarthritis to occur.

After the registration of the left knee of the rats with chemical osteoarthritis, their number was divided into 4 groups. Astaxanthin + hyaluronic acid to Group 1, astaxanthin to Group 2, steroid + hyaluronic acid to Group 3 and hyaluronic acid treatments to Group 4 were applied to the left hind knees with OA injection.

Waiting for four weeks after the treatment was expected to take effect. Four weeks later, the rats were re-run on the Catwalk device and the post-treatment values ​​were recorded.

Results

Results are automatically taken from the Catwalk gait analysis system

Conclusion

Catwalk device is a suitable method for the modeling of knee osteoarthritis in rats because it can record and automatically classify many data at the same time and allows comparison.

The monosodiumiodoacetate molecule produces effects similar to degenerative osteoarthritis disease by chondrolysis induced by cartilages, it is an appropriate method to create an experimental osteoarthritis model.

The astaxanthin molecule, which is in the experimental phase and which has a strong antioxidant properties in the treatment group, is a method that can be used as an alternative to the hyaluronic acid and steroid treatments currently in use in the treatment.

Purpose

The mechanisms of PTOA following anterior cruciate ligament (ACL) injury are likely due to the biological and mechanical insults at the time of injury. These patients still remain at high risk for PTOA after treated with ACL reconstruction (ACL-R). We do not know if the cartilage degeneration after ACL-R is primarily due to abnormal mechanics, inflammation or both. “Idealized” ACLR model (restore normal joint kinematic) was used to test if inflammatory factors are responsible for PTOA.

Methods and Materials

Eighteen-month-old female mini-pigs were divided into “idealized” anterior cruciate ligament reconstruction (IACLR) group and control group (n=16 limbs per group). Real-time PCR, safranine O staining and indian ink staining were performed to verify whether animal models were successfully established or not. Multiple linear regression analysis was used to evaluate the correlation between levels of the inflammatory factors by the Luminex (including interferon [IFN]-γ, interleukin [IL]-1β, IL-4, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor [TNF]-α) and changes in cartilage histology (quantified by morphological scoring) after surgery.

Results

A significant OA cartilage damage with increased MMP-1, MMP-13 mRNA levels and reduced the level of aggrecan mRNA/protein was observed in IACLR groups. Gross morphology score from IACLR animals was dramatically increased compared with control. Moreover, IACLR animals significantly increased the levels of IL-1β, IL-4, IL-6 and TNF-α in the synovial fluid of the knee compared with control. The levels of IL-1β, IL-6 and TNF-α are correlated with morphological score of PTOA.

Conclusion

IACLR is well-known restoring normal joint kinematic model. These results demonstrated that inflammatory factors are independently responsible for the onset of PTOA.

Purpose

The synovial membrane lines the inner surface of the joint cavity and has an important role in controlling the synovial microenvironment. Recent studies show that synovitis promotes arthritic disease progression in the knee [1], however little is known on the role of the synovial membrane in aiding or hindering tissue regeneration following cartilage repair therapy. This study aims to evaluate the response of the synovial membrane to bone marrow stimulation (BMS) by microdrilling of a chronic cartilage defect [2].

Methods and Materials

Following ethics-approved protocols, 4x4 mm unilateral or bilateral cartilage defects were created in the femoral trochlea of skeletally mature NZW rabbits (N=9), brought to chronicity over four weeks, and then microdrilled with four 0.9 mm diameter holes at 2 mm depth. Supra-patellar synovial membranes were collected 1 week and 3 weeks after BMS. Starting at 1-week post-BMS, N=6 rabbits were submitted to 3 weekly intra-articular injections of saline or Synvisc®. Synovial membranes were analyzed for lining thickness, blood vessel density by stereology and macrophages infiltration by histomorphometry.

Results

Following a small medial arthrotomy and BMS, the supra-patellar synovial membrane exhibits typical signs of synovitis. The synovial lining was significantly thicker at 1-week and at 3-weeks post-operative compared to the synovium of intact knees (Fig. 1). RAM-11+ macrophage infiltration increased progressively through 3 weeks (Fig. 2A), but recruitment of wound-healing M2 macrophages to the synovial subintima was the highest at 1-week then diminished after 3-weeks of saline or Synvisc® IA-injection (Fig. 2B).

Conclusion

Following BMS, the supra-patellar synovial membrane displayed a strong inflammatory response similar to synovitis and was sustained up to 3-weeks post-operative. This response could be required or detrimental to cartilage repair processes and requires further study.

Acknowledgements: We thank Jun Sun. Funding sources: CIHR, NSERC.

References: [1] Sellam (2010) Nat Rev Rheumatol; [2] Dwivedi (2019) J Tissue Eng Regen Med.

Purpose

Osteoarthritis (OA) is the most common chronic degenerative joint disease, causing pain and decreased functionality. MSCs from adipose tissue (ADSCs) are attractive stem cell types thanks to their abundance, ease of harvest and high proliferation potential. ADSCs can be used as cell suspension (SVF) (one-step) or in vitro expanded (two-step). Preclinical studies yet showed the potential of expanded ADSCs in inhibiting OA progression. The amniotic epithelial stem cells (AECs) have regenerative properties similar to those of embryonic ones, are rapidly available, have not ethical concerns and don’t form tumor after transplantation. Currently, no authors evaluated AEC effects in cartilage diseases. This study aims to develop a new minimally invasive biological approach for OA treatment, comparing the therapeutic potential of AECs, ADSCs and SVF in the treatment of experimentally induced OA in sheep.

Methods and Materials

24 adult female sheep underwent to bilateral, lateral meniscectomy accordingly to Delling et al. The 48 knee joints randomly received an intra-articular injection of expanded allogenic AECs, autologous SVF, autologous expanded ADSCs or saline solution (control), 6 weeks after meniscectomy. After 3 and 6 months from the treatments, joints were evaluated with macroscopy, histology and histomorphometry.

Results

From the preliminary macroscopic evaluations (Figure 1), it was shown that: 1) lateral compartment (red circles) was the most affected by OA; 2) the highest OA signs were observed in the control condyles, especially at 6 months; 3) the best infiltrative treatments were SVF or AEC injections; 4) worst results were obtained with culture expanded ADSCs.

Conclusion

Bilateral, lateral meniscectomy induces OA that gets worse over time. One injection of SVF or AECs, 6 weeks after OA induction, seems to improve the clinical picture, while 1 injection of culture expanded ADSCs doesn’t ameliorate OA signs. These preliminary results will be confirmed by histology and histomorphometry that are in progress.

Purpose

We sought to investigate the mechanism of epidermal growth factor receptor (EGFR) in regulating osteoarthritis (OA), and identify a drug which could restore the balance between cartilage matrix synthesis and degradation in the corresponding OA subpopulation.

Methods and Materials

Immunohistochemistry was performed to evaluate EGFR activation in the cartilage of OA patients. The conditional gene knockout (Col2a1-creER^T2; Egfr^f/f) mice were utilized and the destabilization of medial meniscus (DMM) surgery was conducted to induce OA. Gefitinib encapsulated chitosan microspheres or lentivirus were injected into the joints for treatment or gene knockdown. Safranin O and immunostaining were performed to evaluate EGFR activation and OA progression. For in vitro study, qPCR and western blot analysis were utilized for transcriptional and translational change detection respectively. RNA sequencing was also performed for the transcriptome alteration analysis.

Results

The heterogeneous activation of EGFR among the OA patients was discovered, and the subpopulation of OA patients displaying high activation level of EGFR (indicated by phosphorylated EGFR) was identified. With Col2a1-creER^T2; Egfr^f/fmice, it was found that EGFR inhibition prohibited cartilage matrix degeneration and promoted cartilage regeneration in the mouse OA model. Consistent with the clinical case report, The Food and Drug Administration (FDA)-approved drug gefitinib could efficiently inhibit EGFR functions to restore chondrocyte phenotype in vitro and preserve OA joints in vivo. Mechanism study showed that gefitinib exerts its protective effect through re-activating autophagy in the chondrocytes.

Conclusion

Our findings suggested the concept of the EGFR activated OA subpopulation and illustrated the mechanism of EGFR signaling in regulating cartilage homeostasis. Gefitinib could be a promising disease-modifying drug for this OA subpopulation treatment.

Purpose

To evaluate the effects of TRB-N0224, a chemically modified curcumin with zinc binding properties and improved pharmacokinetics, in a rabbit injury-induced model of osteoarthritis (OA).

Methods and Materials

38 skeletally mature New Zealand-like rabbits were studied in 4 groups: a sham with arthrotomy (n=6), control with ACL transection (n=6), and two treatment groups with ACL transection and administration of TRB-N0224 at low (25mg/kg/day) (n=13) and high (50mg/kg/day) (n=13) doses. After euthanization at 12 weeks, outcomes were measured by post-necropsy gross morphology, biomechanics, and cartilage and synovium histology. Rabbit blood ELISA quantified cytokine and MMP concentrations at 0, 4, 8, and 12 weeks.

Results

Both treatment groups had fewer distal femoral condyle defects than the control; the low dose demonstrated a mean 78% decrease (p<0.01). Biomechanical testing of the tibial femoral condyle revealed 1.41 MPa lower compressive strength in treatment groups than the control (p<0.05). This finding did not correlate with other tested parameters. According to the Mankin scale for OA, the low and high dose cartilage had fewer histopathological changes exhibited by lower scores (2.60 and 1.25, respectively) than the control (3.50). The control had more severe synovitis than both treatment groups. ELISA results suggested that the key mediators of OA, IL-1β, IL-6, TNFα, MMP-9, and MMP-13, decreased concentrations with TRB-N0224 treatment between weeks 4 to 12.

Conclusion

In pathogenesis of OA, an imbalance exists between catabolic and anabolic mediators. These results suggest the potential of TRB-N0224 to modulate MMPs and cytokines, the catabolic mediators, slowing the macroscopic and histopathological progression of OA.