Homing and reparative effect of intra-articular injection of autologus mesenchymal stem cells in osteoarthritic animal model

Abir N Mokbel, Omar S El Tookhy, Ashraf A Shamaa, Laila A Rashed, Dina Sabry, Abeer M El Sayed, Abir N Mokbel, Omar S El Tookhy, Ashraf A Shamaa, Laila A Rashed, Dina Sabry, Abeer M El Sayed

Abstract

Background: This work aimed to study the homing evidence and the reparative effect of mesenchymal stem cells (MSCs) in the healing process of induced osteoarthritis in experimental animal model (donkeys).

Methods: Twenty-seven donkeys were equally divided into 3 groups based on the observation period after induction of arthritis (3, 6 and 9 weeks) to achieve different degrees of osteoarthritis. Each group was subdivided into three subgroups of three animals each based on the follow-up period (1, 2 and 6 months) after treatment. The induction was done through intra-articular (IA) injection of 2 ml of Amphotericin-B in both carpal joints. MSCs were harvested in a separate procedure, labeled with green fluorescent protein (GFP) using monster GFP vector and suspended in hyaluronic acid for IA injection. Treatment approaches consisted of cell-treatment using MSCs suspended in 3 ml of hyaluronic acid (HA) for the right carpal joint; and using the same amount of (HA) but without MSCs for the left contralateral carpal joint to serve as a control. Animals were assessed clinically and radiologically before and after treatment. Synovial fluid was also evaluated. Histopathologically; articular cartilage structural changes, reduction of articular cartilage matrix staining, osteophyte formation, and subchondral bone plate thickening were graded. Data was summarized using median and percentile for scores of histopathologic grading. Comparison between groups was done using non-parametric Mann Whitney test.

Results: The reparative effect of MSCs was significant both clinically and radiologically in all treated groups (P < 0.05) compared to the control groups. Fluorescence microscopy of sections of the cell-treated joints of all animals indicated that the GFP-transduced injected cells have participated effectively in the reparative process of the damaged articular surface and have integrated within the existing articular cartilage. The cells were associated with the surface of the cartilage and, were also detected in the interior.

Conclusions: Homing was confirmed by the incorporation of injected GFP-labeled MSCs within the repaired newly formed cartilage. Significant recovery proves that the use of IA injection of autologous MSCs is a viable and a practical option for treating different degrees of osteoarthritis.

Figures

Figure 1
Figure 1
Schematic diagram of the animal groups showing the induction of different degrees of arthritis with the following observation period, then the treatment stage with the follow up period.
Figure 2
Figure 2
Flow cytometric characterization analyses of bone marrow-derived MSCs. Cells were uniformly negative for CD34, and positive for CD29.
Figure 3
Figure 3
(a) MSCs-BM cells in culture without adding growth factors for osteogenic and chondrogenic differentiation arrows show fibroblast-like cells in morphology. (b) MSCs-BM cells in culture after adding growth factors for osteogenic and (c) chondrogenic differentiation arrows show change in MSCs morphology.
Figure 4
Figure 4
(a) Control undifferentiated MSCs showed neither staining with Alzarin red (special stain for differentiated MSCs into osteoblasts) nor Alcian blue (special stain for differentiated MSCs into chondrocytes); (b) Osteogenic differentiation of these cells shows the presence of alizarin stained calcium deposits in MSCs-BM; (c) Chondrogenic differentiation of MSCs-BM shows the presence of Alcian blue stained of differentiated cells. All these images are at a magnification of 20X.
Figure 5
Figure 5
Anterio-posterior radiographic image of the carpal joint, showing different arthritic changes in the untreated joints manifested by mild, moderate and severe narrowing of the joint spaces at (a) 1 month, (b) 2 month and (c) 6 month respectively, post injection of Amphotericin-B.
Figure 6
Figure 6
Latro-medial radiographic image of the carpal joint, showing Different arthritic changes manifested by mild, moderate and severe narrowing of the joint spaces at (a) 1 month, (b) 2 month and (c) 6 month respectively, post injection of Amphotericin-B.
Figure 7
Figure 7
Flexed radiographic image of the carpal joint of group-III, showing the cartilage at (a) 1 month, (b) 2 month and (c) 6 month post injection of Amphotericin-B . Notice the thining of the articular cartilage compared to treated carpal joints -images on the right- at (d) 1 month, (e) 2 month and (f) 6 month post treatment with MSCs. Compare arrow with its contralateral.
Figure 8
Figure 8
Histograms showing physical, cytological and biochemical characteristics of synovial fluid analysis of the control and experimental groups.
Figure 9
Figure 9
(a) left antebrachiocarpal joint after 9 weeks of OA induction and 6 month of no treatment showing severe articular surface degeneration affecting the 1: ulnar carpal bone, 2: intermediate carpal bone and 3: radial carpal bone. Notice the degenerative line marked by the arrow; (b) right antebrachiocarpal joint after 9 weeks of OA induction and 6 month of MSCs treatment showing improved articular surface. Notice the partial degenerative areas marked by the arrows; (c) left untreated joint of group-I after 2 month follow-up and (d) right treated joint; (e) untreated joint of group-II after 6 month follow-up (f) treated joints. Arrows indicates areas of degeneration. Compare left images (non-cell-treated) with the contralateral (cell-treated) ones.
Figure 10
Figure 10
Fluorescence microscopic analysis of the cell-treated joints showing GFP-positive cells detected at the surface and also in the center of regenerated tissue in all groups. Group-I; (a) at 2 months and (b) at 6 month after injection of GFP-transduced MSCs. Group-II; show diffuse hypercellularity after 2 months (c) and clusters of chondrocytes after 6 months of injection (d). Group-III; show slight hypercellularity after 2 month (e) with clusters of chondrocytes (short arrow) and multiple tide marks (long arrow) after 6 months of injection (f).
Figure 11
Figure 11
Articular cartilage of group-I two months post injection: (a) control joint showing degenerative changes in the form of hypocellularity, fibrillation (arrow) & fissures (dotted arrows) (H&E 200X); (b) MSCs treated joint showing regenerative changes in articular cartilage including diffuse hypercellularity chondrocyte clones (arrows), regular surface and moderate decrease in matrix staining in superficial and middle zones (Alcian blue-PAS 100X). Articular cartilage of group-I six months post injection: (c): control joint showing duplicated tide marks (arrows) and moderate thickening of subchondral bone plate (H&E 100X); (d) MSCs treated joints showing regenerative changes in the form of moderate decrease of staining intensity of extracellular matrix with hypercllularity (MT 100X).
Figure 12
Figure 12
Articular cartilage of group-II one month post injection: (a) control joint showing superficial fibrillation & clefts involving 1/3 with slight hypocellularity (H&E 100X); (b): MSCs-treated joint showing hypercellularity and marked decrease in staining with focal areas showing synthesis of extracellular matrix (arrows) (MT 100x). Articular cartilage of group-II two months post injection: (c) control joint showing irregular surface with superficial fibrillation & clefts involving up to 2/3 with slight hypercellularity and moderate subchondral bone thickening (H&E 100x); (d)MSCs, treated joint showing regenerative changes in articular cartilage with slight hypercellularity and increased matrix synthesis (arrows) in lower zone (Alcian blue-PAS 100x). Articular cartilage of group-II six months post injection: (e) control joint showing near total replacement of articular cartilage with fibrous tissue, loss of chondrocytes and marked subchondral bone thickening (H&E 100x); (f) MSCs-treated joint showing degenerative changes in the form of irregular surface, fissures and hypocellularity in superficial & middle zone and regenerative changes in lower zone denoted by increased matrix synthesis (arrows) (Alcian blue-PAS 100x).
Figure 13
Figure 13
Articular cartilage of group-III one month post injection: (a) control joint showing surface irregularity with hypocellularity and marked multiple tide marks (H&E 100x); (b) MSCs- treated joint showing degenerative changes with irregular surface, erosion (arrow) and hypocellularity in superficial zone and regenerative changes in middle zone & lower zone denoted by increased matrix synthesis, slight hypercellularity and chondrocytes clones (Alcian blue-PAS 100x). Articular cartilage of group-III two months post injection: (c) control joint showing surface erosion of articular cartilage with superficial loss of chondrocytes and moderate hypercellularity of rest of cartilage (H&E 100x); (d) MSCs-treated joint showing degenerative changes in the form of irregular surface, hypocellularity in superficial & middle zone and regenerative changes in lower zone denoted by hypercellularity& increased matrix synthesis (Alcian blue-PAS 100x). Articular cartilage of group-III six months post injection: (e) control joint showing articular cartilage destruction, pannus formation (arrow) and marked subchondral plate thickening (H&E 100x); (f) MSCs-treated joint showing articular cartilage destruction, fibrous tissue, scattered atrophic chondrocytes and marked subchondral plate thickening (H&E 100x).

References

    1. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. Journal of Cellular Biochemistry. 2006;98(5):1076–1084. doi: 10.1002/jcb.20886.
    1. Sordi V. Mesenchymal Stem Cell Homing Capacity. Transplantation. 2009;87(9S):S42–S45.
    1. Barry FP. Biology and clinical applications of mesenchymal stem cells. Birth Defects Res C Embryo Today. 2003;69(3):250–6. doi: 10.1002/bdrc.10021.
    1. Nöth U, Steinert AF, Tuan RS. Technology Insight: adult mesenchymal stem cells for osteoarthritis therapy: Delivery modes for Mesenchymal stem cells. Nature Clinical Practice Rheumatology. 2008;4:371–380.
    1. Aigner T, Soder S, Gebhard P, McAlinden A, Haag J. Mechanisms of disease: role of chondrocytes in the patho- genesis of osteoarthritis--structure, chaos and senescence. Nat Clin Pract Rheumatol. 2007;3:391–399. doi: 10.1038/ncprheum0534.
    1. Di Cesare PE, Abramson SB, Samuels J. In: Pathogenesis of Osteoarthritis. Firestein GS, Firestein et al, editor. Kelley's Textbook of Rheumatology, 8E, (Chapter 89), Philadelphia; 2008.
    1. Ameye LG, Young MF. Animal Models of Osteoarthritis: Lessons Learned While Seeking the 'Holy Grail'. Curr Opin Rheumatol. 2006;18(5):537–547. doi: 10.1097/.
    1. Bowman KF, Purohit RC, Ganjam VK, Pechman RD Jr, Vaughan JT. Thermographic evaluation of corticosteroid efficacy in Amphotericin-B -induced arthritis in ponies. Am J Vet Res. 1983;44:51–56.
    1. Crawford WH, Houge JC, Neirby DT, Di Mino A, Di Mino AA. Pulsed radio frequency therapy of experimentally induced arthritis in ponies. Can J Vet Res. 1991;55:76–85.
    1. Fahmy AS, Hegazy AA, Abdelhamied MA, Shamaa AA, Schimke E. Clinical, Biochemical and Histopathological studies on arthritis in equine. Vet Med J Giza. 1994;42(1):305–320.
    1. Hegazy AA, Fahmy LS, Fahmy AS, Abdelhamied MA, Shamaa AA, and Schimke E. Evaluation of the effects of intra-articular injection of dimethylsulfoxide on chemically induced arthritis in equines. Vet Med J Giza. 1994;42:221–243.
    1. Suominen MM, Tulamo RM, Puupponen LM, Sankari SM. Effects of intra-articular injections of bufexamac suspension on Amphotericin-B -induced aseptic arthritis in horses. Am J Vet Res. 1999;60(12):1467–73.
    1. Marttinen PH, Raulo SM, Suominen MM, Tulamo RM. Changes in MMP-2 and -9 activity and MMP-8 reac- tivity after Amphotericin-B induced synovitis and treatment with bufexamac. J Vet Med. 2006;53:311–318. doi: 10.1111/j.1439-0442.2006.00837.x.
    1. Shamaa A, Mokbel A, El-Tookhy O, Mostafa A. The efficiency of Intra-articular injection of Amphotericin-B in inducing arthritis in experimental Equine model. Accepted at 11th Scientific conference (3rd International) 15-18, May 2011, Faculty of Veterinary Medicine, Cairo University;
    1. Jiang W, Ma A, Wang T, Han K, Liu Y, Zhang Y, Dong A, Du Y, Huang X, Wang J, Lei X, Zheng X. Homing and differentiation of mesenchymal stem cells delivered intravenously to ischemic myocardium in vivo: a time-series study. European Journal of Physiology. 2006;453(1):43–52. doi: 10.1007/s00424-006-0117-y.
    1. Kollar K, Cook MM, Atkinson K, Brooke G. Molecular Mechanisms Involved in Mesenchymal Stem Cell Mi- gration to the Site of Acute Myocardial Infarction. International Journal of Cell Biology. 2009;2009:904682. pages 8.
    1. Syková E, Jendelová P, Urdzíková L, Lesný P, Hejcl A. Bone marrow stem cells and polymer hydrogels-two strategies for spinal cord injury repair. Cell Mol Neurobiol. 2009;26(7-8):1113–29.
    1. Hu SL, Luo HS, Li JT, Xia YZ, Li L, Zhang LJ, Meng H, Cui GY, Chen Z, WuN, Lin JK, Zhu G, Feng H. Functional recovery in acute traumatic spinal cord injury after transplantation of human umbilical cord mesenchymal stem cells. Critical Care Medicine. 2010;38(11):2181–2189. doi: 10.1097/CCM.0b013e3181f17c0e.
    1. Miller RH, Bai L, Lennon DP, Caplan AI. The Potential of Mesenchymal Stem Cells for Neural Repair. Discovery Medicine. 2010;9(46):236–242.
    1. Murphy MJ, Fink DJ, Hunziker EB, Barry FP. Stem cell therapy in a Caprine model of osteoarthritis Arthritis & Rheumatism. 2003. pp. 3464–3474.
    1. El-Tookhy O, AbouElkheir W, Mokbel A, Osman A. Intra-articular Injection of Autologous Mesenchymal Stem Cells in Experimental Chondral Defects in Dogs. Egypt Rheumatologist. 2008;30(2):1–10.
    1. American Psychological Association. Guidelines for Ethical Conduct in the Care and Use of Animals. 1996.
    1. Anon J. Guide for Veterinary Service and Judging of Equestrian Events. 4. Lexington KY: Am Assoc Equine Practnr; 1991.
    1. Martin A, Sandra O, Mandi J, Daniel B, Olga B, Jill R, Rustin M, Jeffrey M. Comparison of Chondrogenic Potential in Equine Mesenchymal Stromal Cells Derived from Adipose Tissue and Bone Marrow. Vet Surg. 2008;37(8):713–724. doi: 10.1111/j.1532-950X.2008.00462.x.
    1. Radcliffe CH, Flaminio MJ, Fortier LA. Temporal Analysis of Equine Bone Marrow Aspirate During Establishment of Putative Mesenchymal Progenitor Cell Populations. 2. Vol. 19. Stem cells and Development; 2010.
    1. Zhi-Yong Z, Swee-Hin T, Mark S, Jan T, Nicholas M, Mahesh A, Jeery C. Superior Osteogenic Capacity for Bone Tissue Engineering of Fetal Compared with Perinatal and Adult Mesenchymal Stem Cells. Stem cells. 2009;27:126–137. doi: 10.1634/stemcells.2008-0456.
    1. Niki H, Hosokawa S, Nagaike K, Tagawa T. A new immunofluorostaining method using red fluorescence of PerCP on formalin-fixed paraffin-embedded tissues. J Immunol Methods. 2004;293(1-2):143–51. doi: 10.1016/j.jim.2004.07.009.
    1. Maurisse R, De Semir D, Emamekhoo H, Bedayat B, Abdolmohammadi A, Parsi H, Gruenert D. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnology. 2010;10(9)
    1. Eronen I, Videman T, Friman C, Michelsson JE. Glycosaminoglycan metabolism in experimental osteoarthrosis caused by immobilization. ActaOrthop Scand. 1978;49(4):329–34.
    1. Carlson CS, Guilak F, Vail TP, Gardin JF, Kraus VB. Synovial fluid biomarker levels predict articular cartilage damage following complete medial meniscectomy in the canine knee. J Orthop Res. 2002;20:92–100. doi: 10.1016/S0736-0266(01)00066-3.
    1. Murphy JM, Dixon K, Beck S, Fabian DF, Feldman A, Barry FP. Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis Rheum. 2002;46:704–13. doi: 10.1002/art.10118.
    1. Kotschwar JL, Coetzee JF, Anderson DE, Gehring R, KuKanich B, Apley MD. Analgesic efficacy of sodium salicylate in an Amphotericin-B -induced bovine synovitis-arthritis model. Dairy Sci. 2009;92:3731–3743. doi: 10.3168/jds.2009-2058.
    1. Appleton CTG, McErlain DD, Pitelka V, Schwartz N, Bernier SM, Henry JL, Holdsworth DW, Beier F. Forced mobilization accelerates pathogenesis: characterization of a preclinical surgical model of osteoarthritis. Arthritis Res Ther. 2008;10:407. doi: 10.1186/ar2513.
    1. Ayotte R, Laurin CA. Pathogenesis of joint effusions: Anexperimental study. Can Med A. 1969;100:242–250.
    1. McIlwraith CW, Fessler JF, Blevins WE, Page EH, Rebar AH, Van Sickle DC, Coppoc GL. Experimentally induced arthritis of the equine carpus: clinical determinations. Am J Vet Res. 1979;40(1):11–19.
    1. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy. 2006;8:315–317. doi: 10.1080/14653240600855905.
    1. Tatebe M, Nakamura R, Kagami H, Okada K, Ueda M. Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit. Cytotherapy. 2005;7(6):520–530. doi: 10.1080/14653240500361350.
    1. Ueng SW, Yuan LJ, Lin SS, Liu SJ, Chan EC, Chen KT, Lee MS. In vitro and in vivo analysis of a biodegradable poly (lactide-co-glycolide) copolymer capsule and collagen composite system for antibiotics and bone cells delivery. J Trauma Jun. 2011;70(6):1503–9. doi: 10.1097/TA.0b013e3181edb873.
    1. Koga H, Shimaya M, Muneta T, Nimura A, Morito T, Hayashi M, Suzuki S, Ju YJ, Mochizuki T, Sekiya I. Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect. Arthritis Res Ther. 2008;10(4):R84..
    1. Mankin HJ, Dorfman H, Lipiello N, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips II: Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am. 1971;53-A:523–537.
    1. Melo EG, Gomes MG, Nunes VA, Rezende CMF. Effects of chondroitin sulfate and sodium hyaluronate on chondrocytes and extracellularmatrix of articular cartilage in dogs with degenerative joint disease. Arq Bras Med Vet Zootec. 2008;60(1):93–102. doi: 10.1590/S0102-09352008000100014.
    1. Smith GN, Myers SL, Brandt KD, Mickler EA. Effect of intraarticular hyaluronan injection in experimental canine osteoarthritis. Arth Rheum. 1998;41:976–985. doi: 10.1002/1529-0131(199806)41:6<976::AID-ART4>;2-R.
    1. Guidolin DD, Ronchetti IP, Lini E, Guerra D, Frizziero L. Morphological analysis of articular biopsies from randomized, clinical study comparing the effects of 500-730 kDa sodium hyaluronate (Hyalgan®) and methylpredinisolone acetate on primary osteoarthritis of the knee. Osteoarth Cart. 2001;9:371–381. doi: 10.1053/joca.2000.0398.
    1. Barbucci R, Lamponi S, Borzachiello A, Ambrosio L, Fini M, Torricelli P, Giardino R. Hyaluronic acid hydrogel in the treatment of osteoarthritis. Biomaterials. 2002. pp. 4503–4513.
    1. Sandell LJ, Aigner T. Articular cartilage and changes in arthritis An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3:107–113. doi: 10.1186/ar148.
    1. McCarty MF. Glucosamine for wound healing. Med Hypotheses. 1996. pp. 273–275.
    1. Chen WY, Abatangelo G. Functions of hyaluronan in wound repair. Wound Repair. 1999;7:79–89. doi: 10.1046/j.1524-475X.1999.00079.x.

Source: PubMed

Patrocinadores y Colaboradores

Condiciones médicas

Intervenciones de drogas

3
Suscribir