This study showed the effectiveness of combination therapy of osteoarthritic synovial derived stem cells (OA-SDSCs) with subchondral drilling for large osteochondral defects repair in mature rabbit model. The defect was created at load-bearing area of the medial femoral condyle of both knees (6 mm length × 3 mm width). Then, mature rabbits were separated randomly into 2 groups: 3 subchondral holes were penetrated the subchondral bone in the defect site (drilled group), and then an intra-articular injection of one million OA-SDSCs into the knee joint was performed (combined group). After two months, rabbits were euthanized to perform histological assessment of the repaired tissue using safranin O stain. Repaired tissue was visually more whitish in the drilled group than in the combined group. Histologically, repaired tissue almost revealed fibrocartilage with subchondral repair in the combined group. However, fibrous tissue was represented in the drilled group. On Pineda score, the combined group was significantly better than of the drilled group (P = 0.001). Finally, using of OA-SDSCs with subchondral drilling promotes better cartilage repair than using subchondral drilling alone

Mahmoud, Elhussein Elbadry

Mawas, Amany Sayed

English

Journal of Advanced Veterinary Research (University Assiut, Egypt)

IntroductionHealing of the articular cartilage lesions is potentially lim-ited because of its poor vascularization. Although bone mar-row stimulation such as subchondral drilling is the mostcommon traditional surgical treatment option for cartilage le-sions (Pridie, 1959). However, its clinical outcome is not satis-factory due to the formation of fibrous tissue or fibrocartilage(Knutsen et al., 2004), which does not resemble the surround-ing hyaline cartilage. The subchondral drilling has aimed toclot formation through blood migration containing mes-enchymal stem cells (MSCs) from the bone marrow into thedefect site (Madry et al., 2010). MSCs have the ability to dif-ferentiate into chondrocytes, osteoblasts, and adipocytes (Pit-tenger et al., 1999), which participate in fibrocartilageformation due to their low concentration in the bone marrow. Most of the previous studies have used monolayer cul-tured bone marrow MSCs with subchondral drilling to pro-mote osteochondral repair than using subchondral drillingalone in rats (Nishimori et al., 2006), and goats (Nam et al.,2013). A new source of MSCs from synovial tissue hasrecorded, and named synovial MSCs. These cells are a goodoption for cartilage repair because of their high proliferativecapacity and chondrogenic potential (Sakaguchi et al., 2005),and easy harvesting synovial tissue arthroscopically with lowdegree of minimal invasiveness (De Bari et al., 2001). Although,Interleukin (IL)-1β has been shown to trigger pathologicalprocesses in rheumatoid arthritis and osteoarthritis (OA), theproliferation rate and chondrogenic potency of synovial MSCswere improved after treatment with IL-1β (Matsumura et al.,2017). For this reason, we hypothesized that osteoarthriticsynovial derived stem cells (OA-SDSCs) have the potential toenhance chondrogenic capacity, maintain proliferative prop-erty, and have improved quality of the repaired articular car-tilage. Hence, this study was aimed to examine the impact ofan intra-articular injection of OA-SDSCs with subchondraldrilling for large osteochondral defects repair in of the maturerabbit model.  Materials and methodsThis study was approved by ethical committee of researchfacility (Faculty of Veterinary Medicine, South Valley University,Egypt.*Corresponding author: Elhussein Elbadry MahmoudE-mail address: E.badry@vet.svu.edu.egJournal of Advanced Veterinary Research Volume 10, Issue 1 (2020) 13-16Journal of Advanced Veterinary Researchhttps://advetresearch.com1Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt. 2Department of Surgery, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.ABSTRACTISSN: 2090-6277/2090-6269/ © 2011-2020 JAVR. All rights reserved.Osteoarthritic Synovial Derived Stem Cells Augmented with SubchondralDrilling for Repair of Large Osteochondral Defects in Rabbit ModelAmany Sayed Mawas1, Elhussein Elbadry Mahmoud2*Abstract: This study showed the effectiveness of combination therapy of osteoarthritic synovial derivedstem cells (OA-SDSCs) with subchondral drilling for large osteochondral defects repair in mature rabbitmodel. The defect was created at load-bearing area of the medial femoral condyle of both knees (6mm length × 3 mm width). Then, mature rabbits were separated randomly into 2 groups: 3 subchondralholes were penetrated the subchondral bone in the defect site (drilled group), and then an intra-articularinjection of one million OA-SDSCs into the knee joint was performed (combined group). After twomonths, rabbits were euthanized to perform histological assessment of the repaired tissue usingsafranin O stain. Repaired tissue was visually more whitish in the drilled group than in the combinedgroup. Histologically, repaired tissue almost revealed fibrocartilage with subchondral repair in the com-bined group. However, fibrous tissue was represented in the drilled group. On Pineda score, the com-bined group was significantly better than of the drilled group (P = 0.001). Finally, using of OA-SDSCswith subchondral drilling promotes better cartilage repair than using subchondral drilling alone.ARTICLE INFOOriginal ResearchReceived:07 November 2019Accepted:21 December 2019Keywords:Subchondral drilling,OA-SDSCs, Osteochondralrepair, Rabbit modelJ. Adv. Vet. Res. (2020), 10 (1), 13-16Isolation and expansion of OA-SDSCsThe synovial tissue was collected from the medial aspectof the osteoarthritic knee joint of the rabbit for isolation ofOA-SDSCs using explant method. In brief, synovial tissue wasdivided into tiny pieces and cultured on culture dishes, andOA-SDSCs started to migrate from the periphery of the tissueand strongly adhered to culture dishes to form colonies. TheseOA-SDSCs were cultured in Dulbecco's Modified EagleMedium (DMEM) (Gibco®, Life Technologies, USA) containing10% deactivated fetal bovine serum (FBS) (Sigma-Aldrich, St.Louis, MO) and 1% antibiotics (penicillin and streptomycin)(Invitrogen, CA, USA). Then, the tissue was removed after 1week. Cells were seeded on 10-cm2 tissue culture dishes (BDFalcon™; BD Biosciences, Franklin Lakes, NJ), and incubated ina humidified atmosphere with 5% Co2 at 37˚C. After 2 weeks,0.25% trypsin (Gibco®, Thermo Fisher Scientific, Waltham,MA) was used for 2 minutes for cell detachment, the cells weresubcultured under the same conditions. These adherent cellsat first passage are referred to as OA-SDSCs that were used inthis experiment (Mahmoud et al., 2019b).Intra-articular injection of OA-SDSCs into the knee jointIn the present study, ten male white rabbits, above 8months of age and weighing 3.0-3.49 kg, were kept in sepa-rate cages freely. An intravenous infusion of pentobarbital (30mg/kg body weight) was performed for induction of anesthe-sia (Mahmoud et al., 2019a). With lateral patellar dislocation,medial parapatellar surgical approach was used to expose theknee joint. Using an electric drill, bilateral osteochondral de-fects (Length 6 mm; width 3 mm) were created on the weight-bearing portion of the medial femoral condyles. Then the 20rabbits' knees were randomly divided into 2 groups equally:Group 1 (the drilled group), three holes were drilled for sub-chondral perforation by 0.5 mm Kirschner wire (n=10). How-ever, in group 2 (the combined group), three holes were drilledfor subchondral perforation with an intra-articular injection of1×106 OA-SDSCs (n.=10), then the joint capsule was closed inroutine manner.Macroscopic and histological assessment of the repaired tissueAt 2 months post-treatment, rabbits were euthanized byintravenous injection of a lethal dose of pentobarbital (180mg/kg body weight). The femoral condyles were collected formacroscopical evaluation, fixed in a 4% paraformaldehydephosphate buffered solution (PFA) for 48 hours, and then de-calcified with 10% ethylenediaminetetraacetic acid solution(EDTA) (Nacalai Tesque, Inc.) for 10 weeks. The samples wereprepared in paraffin sections and cut into 5-µm-thick sagittalsections which were stained with Safranin O/fast green stainfor histological scoring using Pineda score graded with 0 pointas the best and 14 points as the worst (Table 1) (Pineda et al.,1992). Statistical analysisThe histological scoring was analyzed by using of Mann-Whitney' U-test, with a 95% confidence interval. Differencesof P < 0.05 were considered significant.14Amany S. Mawas and Elhussein E. Mahmoud /Journal of Advanced Veterinary Research 10 (1) (2020) 13-16Fig. 1. Representative macroscopic findings of right and left knees were indicated a good condition of the reparative tissue in case of combined group whencompared with the drilled group.(I) Filling of defect125% 1100% 075% 150% 225% 30% 4(II) Reconstitution of osteochondral junctionYes 0Almost 1Not close 2(III) Matrix stainingNormal 0Reduced staining 1Significantly reduced staining 2Faint staining 3No stain 4(IV) Cell morphologyNormal 0Mostly hyaline and fibrocartilage 1Mostly fibrocartilage 2Some fibrocartilage but mostly non chondrocytic cells 3Non chondrocytic cells only 4Table 1. Pineda’s semiquantitative score.ResultsMacroscopic findingsIn the drilled group, repair tissue was soft and morewhitish than normal with easily identifying the defect margins.While in the combined group, repair tissue was somewhat re-sembled the normal one with recognizing defect margins (Fig.1).Histological findings and scoringIn the drilled group, fibrous tissue was formed in the de-fects instead of the subchondral bone and cartilage. In thecombined group, complete repair of the subchondral bone atthe same level of the adjacent normal tissue was confirmed,which covered by fibrocartilage containing chondrocytes orhyaline cartilage. In addition, integration of the reparative tis-sue was good with the surrounding native tissue. At 2 months,the histological scoring results were 10.3±0.5 and 7.9±1.5 forthe drilled group and the combined group, respectively. OnPineda score, the histological score was significantly better inthe combined group than in the drilled group (P= 0.001) (Fig.2).  DiscussionThe hypothesis of this study was consistent with the ob-tained results, which revealed that OA-SDSCs augmented sub-chondral drilling for better repair of large osteochondraldefect of the medial femoral condyle in the mature rabbitmodel, although quality of the reparative tissue was not per-fect.Up till now, one of the common surgical technique for car-tilage repair is subchondral drilling leading to the formationof fibrous tissue, or fibrocartilage with inferior quality than na-tive hyaline cartilage by inducing of the bleeding for recruit-ment of bone marrow mesenchymal stromal cells from thesubchondral bone into the defect site (Frisbie et al., 2003). Inthe present study, fibrous tissue was formed in the large os-teochondral defects in case of drilled group, which may be at-tributed to the traumatic inflammatory response, and lessbleeding from subchondral holes leading to carrying lownumber of bone marrow stromal cells, which were not enoughto restore the large defect. So, it is not good option for usingsubchondral drilling alone for repair of large osteochondraldefect. Previous experimental studies for cartilage repair de-pended upon using subchondral drilling with atelocollagenmembrane (Hamanishi et al., 2013), and scaffolds such as chi-tosan (Merchand et al., 2012). But, the coverage of the defectby scaffold inhibited cartilage repair.  In this study, authors tried to improve quality of the pro-ductive repaired tissue through combination therapy of OA-SDSCs with subchondral drilling which confirmed by completerepair of the subchondral bone covered by fibrocartilage con-taining chondrocytes with good integration with the sur-rounding native tissues. More importantly, SDSCs have beensuccessfully isolated and expanded from pathological synovialtissue from RA or OA patients, providing for a good option forcartilage regeneration/repair (Nagase et al., 2008). ConclusionAn intra-articular injection of OA-SDSCs improved qualityof the reparative tissue produced by subchondral drillingalone. Large number of OA-SDSCs may be more effective fortreating large defects. Therefore, future research trials of com-Fig. 2. A. Representative microscopic findings using Safranin O/ Fast green stain. There was a little fibrous tissue inside the defect in case of the drilled group. While complete repair of the subchondral bone covered by Hyaline/fibro-cartilage including chondrocytes presented in case of the combined group (original magnification: 40X). B. On Pineda histological scoring, combined group was significantly histologically better than of the drilled group. *p < 0.05..Amany S. Mawas and Elhussein E. Mahmoud /Journal of Advanced Veterinary Research 10 (1) (2020) 13-1615bination therapies of different densities of OA-SDSCs with sur-gical procedures such as subchondral drilling to form neo-car-tilage like tissues are required with long-term durability. Conflict of interestThe authors declare no conflict of interest.ReferencesDe Bari, C., Dell'Accio, F., Tylzanowski, P., Luyten, F.P., 2001. Multipotentmesenchymal stem cells from adult human synovial mem-brane. Arthritis Rheum. 44, 1928-1942.Frisbie, D.D., Oxford, J., Southwood, L., Trotter, G.W., Rodkey, W.G.,Stedman, J.R., Goodnight, J.L., Mcllwraith, C.W., 2003. Earlyevents in cartilage repair after subchondral bone microfrac-ture. Clin. Orthop. 407, 215-227.Hamanishi, M., Nakasa, T., Kamei, N., Kazusa, H., Kamei. G., Ochi, M.,2013. Treatment of cartilage defects by subchondral drillingcombined with covering with atelocollagen membrane in-duces osteogenesis in a rat model. J. Orthop. Sci. 18, 627-635.Knutsen, G., Engebretsen, L., Ludvigsen, T.C., Drogset, J.O., Grontvedt,T., Solheim, E., Strand, T., Roberts, S., Isaksen, V., Johansen, O.,2004. Autologous chondrocyte implantation compared withmicrofracture in the knee. A randomized trial. J. Bone JointSurg. Am. 86, 455-464.Madry, H., van Dijk, C.N., Mueller-Gerbl, M., 2010. The basic scienceof the subchondral bone. Knee Surg. Sports Traumatol.Arthrosc. 18, 419-433.Mahmoud, E.E., Adachi, N., Mawas, A.S., Deie, M., Ochi, M., 2019a.Multiple intra-articular injections of allogeneic bone marrow-derived stem cells potentially improve knee lesions resultingfrom surgically induced osteoarthritis: an animal study. BoneJoint J. 101-B, 824-831.Mahmoud, E.E., Adachi, N., Mawas, A.S., Gaarour, O.S., Ochi, M., 2019b.Coculturing of mesenchymal stem cells of different sourcesimproved regenerative capability of osteochondral defect inthe mature rabbit: An in vivo study. J. Orthop. Surg. (HongKong) 27, 2309499019839850.Matsumura, E., Tsuji, K., Komori, K., Koga, H., Sekiya, I., Muneta, T.,2017. Pretreatment with IL-1β enhances proliferation andchondrogenic potential of synovium-derived mesenchymalstem cells. Cytotherapy 19, 181-193.Merchand, C., Chen, G., Tran-Khanh, N., Sun, J., Chen, H., Buschmann,M., Hoemann, C.D., 2012. Microdrilled cartilage defectstreated with thrombin-solidified chitosan/blood implant re-generate a more hyaline, stable, and structurally integratedosteochondral unit compared to drilled controls. Tissue Eng.Part A 18, 508-519.Nagase, T., Muneta, T., Ju, Y.J., Hara, K., Morito, T., Koga, H., Nimura,A., Mochizuki, T., Sekiya, I., 2008. Analysis of the chondrogenicpotential of human synovial stem cells according to harvestsite and culture parameters in knees with medial compart-ment osteoarthritis. Arthritis Rheum. 58, 1389-1398.Nam, H.Y., Karunanithi, P., Loo, W.C.P., Naveen, S.V., Chen, H.C., Hussin,P., Chan, L., Kamarul, T., 2013. The effects of staged intra-ar-ticular injection of cultured autologous mesenchymal stromalcells on the repair of damaged cartilage: a pilot study incaprine model. Arthritis Res. Ther. 15, R129.Nishimori, M., Deie, M., Kanaya, A., Exham, H., Adachi, N., Ochi, M.,2006. Repair of chronic osteochondral defects in the rat. J.Bone Joint Surg. Br. 88-B, 1236-1244.Pineda, S., Pollack, A., Stevenson, S., Goldberg, V., Caplan, A., 1992. Asemiquantitative scale for histologic grading of articular car-tilage repair. Acta. Anat. 143, 335-340.Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R.,Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Mar-shak, D.R., 1999. Multilineage potential of adult human mes-enchymal stem cells. Science 284, 143-147.Pridie, K.H., 1959. A method of resurfacing osteoarthritic knee joints.J. Bone Joint Surg. Br. 41, 618-619.Sakaguchi, Y., Sekiya, I., Yagishita, K., Muneta, T., 2005. Comparison ofhuman stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source. Arthritis Rheum. 52,2521-2529.Amany S. Mawas and Elhussein E. Mahmoud /Journal of Advanced Veterinary Research 10 (1) (2020) 13-1616

Osteoarthritic Synovial Derived Stem Cells Augmented with Subchondral Drilling for Repair of Large Osteochondral Defects in Rabbit Model

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Osteoarthritic Synovial Derived Stem Cells Augmented with Subchondral Drilling for Repair of Large Osteochondral Defects in Rabbit Model

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Journal of Advanced Veterinary Research (University Assiut, Egypt)