Bone Marrow Stromal Stem cells (bMSCs) therapy for musculoskeletal problems (disc, cartilage and bone)


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Bone Marrow Stromal Stem cells (bMSCs) therapy for musculoskeletal problems (disc, cartilage and bone)

  1. 1. <ul><li>Ismail HD * </li></ul><ul><li>Division of Orthopaedic and T raumatology, </li></ul><ul><li>Department of S u rgery, Faculty of Medicine, University Of I ndonesia </li></ul>Bone Marrow Stromal Stem cells (bMSCs) therapy for musculoskeletal problems ( disc, cartilage and bone)
  2. 2. Musculoskeletal problem <ul><li>Disc </li></ul><ul><li>Cartilage </li></ul><ul><li>Bone </li></ul>
  3. 3. Disc and bone Cells matrix
  4. 4. Bone marrow stromal stem cells Skeletal stem cells (SSCs), also known as bone-marrow stromal stem cell, or mesenchymal stem cell
  5. 5. Bone problem TRAUMA NON-TRAUMA FRACTURE ACUTE/FRESH NEGLECTED METABOLIC CONGENITAL ACQUIRED DELAYED/NON-UNION BONE GAPS SHORTENING Limb salvage RECONSTRUCTION in malignancy <ul><li>achondroplasia </li></ul><ul><li>Congenital pseudoarthrosis of tibia </li></ul><ul><li>Coxa vara </li></ul><ul><li>Avascular necrosis </li></ul><ul><li>osteoporosis </li></ul>Fracture repair Reconstruction
  6. 6. Bone Cells matrix <ul><li>Osteoblast </li></ul><ul><li>Osteoclast </li></ul><ul><li>Osteocytes </li></ul><ul><li>Osteoprogenitor cells </li></ul>Organic (40%) Inorganic (60%) Collagen Proteoglycans Noncollagenous : osteocalcin, osteonectin, osteopontin Growth factor and Cytokines : TGF-ß, IGF, IL-1, IL-6, BMP Calsium hydroxyapatite [Ca10(PO4)6(OH)2] Osteocalsium Phosphate (Brushite)
  7. 7. <ul><li>Regardless of the technique, tissue engineering requires three components : </li></ul><ul><ul><ul><li>a growth-inducing stimulus (growth factor) </li></ul></ul></ul><ul><ul><ul><li>responsive cells, and </li></ul></ul></ul><ul><ul><ul><li>a scaffold to support tissue formation </li></ul></ul></ul>
  8. 8. Tissue Engineering Triad
  9. 9. Core concept
  10. 12. <ul><li>factors regulating stem cell self-renewal (Wnts, hedgehog, Dickopf) versus differentiation (BMPs, TGF beta, OP-1)may have to be provided in an appropriate temporal sequence.* </li></ul>Sharp JG. CLINICAL ORTHOPAEDICS AND RELATED RESEARCH .2005;Number 435, pp. 52–61
  11. 13. <ul><li>bMSCs /MSC </li></ul><ul><ul><li>differentiated </li></ul></ul><ul><ul><li>undifferentiated </li></ul></ul>
  12. 14. <ul><li>there is no advantage of implantation of differentiated MSCs. </li></ul><ul><li>  </li></ul><ul><li>Transplantation of mesenchymal stromal cells on mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation </li></ul><ul><li>Authors: Niemeyer, P 1 ; Kasten, P 2 ; Simank, H-G 2 ; Fellenberg, J 2 ; Seckinger, A 3 ; Kreuz, Pc 1 ; Mehlhorn, A 1 ; Südkamp, Np 1 ; Krause, U 3 </li></ul><ul><li>Source: Cytotherapy , Volume 8, Number 4, August 2006, pp. 354-366(13) </li></ul>
  13. 15. <ul><li>In a study of gene therapy targeting stem cells from individuals with osteogenesis imperfecta, the transduced cells were cultured for 2 weeks in bone–differentiation-inducing media before transplantation in vivo to show bone formation. * </li></ul><ul><li>These results imply that in vitro differentiation of stem cell populations can improve bone formation. Unfortunately, this manipulation considerably c omplicates the process of obtaining regulatory approval for clinical application. ** </li></ul><ul><li>Chamberlain JR, Schwarze U, Wang P-R, et al: Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303:1198–1201, 2004 </li></ul><ul><li>** Sharp JG. CLINICAL ORTHOPAEDICS AND RELATED RESEARCH .2005;Number 435, pp. 52–61 </li></ul>
  14. 16. Induction of MSCs to Osteoblast
  15. 18. Characterization of Osteoblasts
  16. 19. <ul><li>mimics the extracellular matrix in regenerating bone environm ent </li></ul><ul><li>Not a simple mechanical support, but has to be ‘informative’ to the cells. </li></ul><ul><li>A proper biomaterial should easily integrate with the adjacent bone and favor new bone tissue ingrowth (osteo-conduction). Its architecture should allow host blood vessels to colonize even the largest structures. </li></ul><ul><li>biocompatible and resorbable. </li></ul><ul><ul><li>platelet-rich plasma (PRP) </li></ul></ul><ul><ul><li>Collagen type 1 </li></ul></ul><ul><ul><li>Hydroxyapatite </li></ul></ul><ul><ul><li>Calsium phosphate </li></ul></ul><ul><ul><li>Polymers : Poly(lactide-co-glycolide) (PLGA) </li></ul></ul>scaffolds
  17. 20. <ul><li>Bone Morphogenic Proteins (BMPs) </li></ul><ul><li>Osteogenic Proteins </li></ul><ul><li>TGF-ß </li></ul>Growth factor
  19. 22. Topic study cells scaffold The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)/carrier complex on bone repair In vitro and in vivo (Sprague-Dawley Rats) {Bone vol 29, no.2, august 2001:169-75} Osteoblastic/BMP Poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS) Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats Animal study (rat) {J Orthop Res 21 (2003) 44-53} MSC engineered with the gene for BMP-2 Collagen gel (vitrogen 100, 95-98% type 1) Calcium phosphate scaffold and bone marrow for bone reconstruction in irradiated area: a dog study animal model (dog) Bone 36 (2005) 323– 330 Bone marrow macroporous biphasic calcium phosphate bone substitute (MBCPk, Biomatlante, Vigneux de Bretagne, France) Treatment of Long Tubular Bone Defect of Rabbit Using Autologous Cultured Osteoblasts Mixed with Fibrin Animal model (New Zealand white rabbits ) J. of Korean Orthopaedic Society Volume 9, Number 1, May, 2006 autologous cultured osteoblasts fibrin
  20. 23. Topic study cells scaffolds Bone Formation of Cultured Osteoblasts in Bone Defect of Radial Shaft of Rabbit Animal model (New Zealand white rabbits ) J. of Korean Orthop. Assoc. 2005; 40: 453-459 autologous cultured osteoblasts The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. non-union defect in adult dog f e mor a J. Bone Joint Surg. Am.1998; 80, 985–996. Autologous MSC hydroxyapatite: beta-tricalcium phosphate (65:35) Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. full-thickness gaps of tibia diaphysis in adult sheep J. Biomed. Mater. Res. 2000;49, 328–337. Autologous bone marrow stromal cells bioceramic composites Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis—a preliminary result of three cases Clinical study achondroplasia patients bilaterally, and three femoral and one tibial lengthenings were done in four patients because of a limb length discrepancy, which was secondary to trauma (two limbs), congenital pseudarthrosis of the tibia (one), and developmental coxa vara (one) Bone 35 (2004) 892– 898 osteoblast-like cells platelet-rich plasma (PRP)
  21. 25. Topic study cells scaffolds Enhanced Tibial Osteotomy Healing with Use of Bone Grafts Supplemented with Platelet Gel or Platelet Gel and Bone Marrow Stromal Cells A prospective, randomized, controlled study . Thirty-three patients undergoing high tibial osteotomy to treat genu varum . J Bone J Surg.Br.2007 ; 11 Osteoblast Platelet gel
  22. 26. Topic study cells scaffolds Treatment of Osteonecrosis of the Femoral Head with Implantation of Autologous Bone-Marrow Cells prospective cohort study. T hirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head . J Bone J Surg.Am. 2004 autologous bone-marrow mononuclear cells
  23. 27. Study by Aziz Nather Department of orthopaedic surgery National University of Singapore
  24. 32. Conclusion 􀂄 Addition of MSCs improved the biological healing of the inert allografts. 􀂄 Addition of PRP did not have the same effect. 􀂄
  25. 33. <ul><li>A major unsolved problem possibly lies in the development of the most appropriate matrix which should be biodegradable yet resistant, permit cell infiltration and adequate survival, proliferation and differentiation of cells, and also provide integration with the pre-existing bony flankin g the lesion sites. </li></ul>
  26. 34. <ul><li>a mixture or a temporal administration of stem and differentiated cell vs undifferentiated populations with matrix and/or growth factors might prove to be the optimal approach. </li></ul>
  27. 35. <ul><li>Disc Problem </li></ul>
  28. 36. Disc Cells matrix Water and proteoglycans increase from outer the annulus to the inner nucleus, and, in contrast, collagens are inversely distributed Collagen type I proteoglycans Nucleus pulposus chondrocyte-like cell Inner annulus Chondrocyte-like cells Outer annulus fibroblast or fibrocyte-like-cells End-plate chondrocyte Proteoglycans : aggrecans, versican, decorin, biglycan, fibromodulin, lumican, and perlecan. Collagen type II
  29. 37. Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc distraction to reverse Intervertebral Disc Degeneration in rabbit model <ul><li>Ismail * Hee Hwan Tak, ** * Nuryati C Siregar, ** James CH Goh,***,Wong Hee Kit, *** Subroto Sapardan * </li></ul><ul><li>Division of Orthopaedic and traumatology, Department of Sirgery, Faculty of Medicine, University Of ndonesia </li></ul><ul><li>** Department of Pathology Anatomy, Faculty Of Medicine, University Of Indonesia </li></ul><ul><li>***Department of Orthopaedic Surgery, National University Hospital, Singapore </li></ul>
  30. 38. Intervertebral Disc Degeneration (IDD) <ul><li>IDD is a multifaceted, chronic process involving certain unfavorable, progressive changes in disc composition, configuration, and function occurring more quickly and or with greater gravity than those associated with normal aging, and often associated with clinical symptoms . </li></ul>
  31. 39. alteration CELL EXTRACELLULER MATRIX Failure of nutrient supply bMSCs <ul><li>Intervertebral disc distraction </li></ul><ul><li>restore disc height </li></ul><ul><li>↑ diffusion </li></ul><ul><li>↑ disc nutrition </li></ul>Regeneration/reparation <ul><li>Differentiated bMSCs can be </li></ul><ul><li>survively transplanted in </li></ul><ul><li>degenerated intervertebral disc </li></ul>
  32. 40. Objectives <ul><li>reverse intervertebral disc degeneration in rabbit model </li></ul>Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
  33. 41. M E T H O D S three sequential stages of experiments were designed :
  34. 42. <ul><li>STAGE 1 : Pilot Study : STUDY TO CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT MODEL </li></ul><ul><li>Intervertebral disc degeneration was created in rabbit model through the application of controlled and quantified axial mechanical loading. </li></ul>
  35. 43. Study on : 1. Developing of external compression and distraction device External compression device External distraction device Stage 2 : Interphase stage
  36. 44. 2. Measuring of cross-sectional area of intervertebral disc of the rabbit L0 L1 Ln-1 Ln Ln + 1 L2 <ul><li>Mean of </li></ul><ul><li>cross-sectional area = 71.21 + 6.4 mm 2 70.1 + 4.61 mm 2 </li></ul>computerized Manual
  37. 45. 3. Establishing of method of isolation, culture, and preparation of transplantation of bone marrow stromal stem cells . Illiac crest bone marrow aspiration from rabbit donor Day 30 x 40
  38. 46. <ul><li>Cultured bMSCs not more than at passage 1 are labeling Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen solution until a final cell density of 1 x 10 6 cells/ml. </li></ul>
  39. 47. In vitro Study : Day 30 : bMSCs embedded in atelocollagen
  40. 48. Stage 3 : intervention stage (bMSCs transplantation and or distraction of the intervertebral disc) <ul><li>Design </li></ul><ul><li>Experimental, post test only control group design </li></ul><ul><li>Location </li></ul><ul><li>Animal Holding Unit, and NUSTEP (National University Tissue Engineering Project), Department of Orthopaedic Surgery, National University Hospital (NUH), and Eijkman Institute, Jakarta, and Faculty of Medicine,University of Indonesia </li></ul><ul><li>. </li></ul>
  41. 49. kept alive for 8 weeks 24 new zealand white rabbits , average weight 3.34 kg Group 1 Group 2 Group 4 Group 3 External compression device, 2.3 MPa, loaded into L4-L5 for 14 days External compression device was removed 8 weeks unload sacrificed bMSCs transplantation Distract ion of the intervertebral disc bMSCs and IVD distraction
  42. 50. Tissue preparation : 22 days Lateral digital X-ray, Micro-CT scan -> disc height Macroscopic : morphological grade (Thomson, et al) Microscopic : histological score (Masuda and Boos,et al) and proteoglycans grade (Norcross, et al) Cell viability (Tunel reaction) and cell labeling (CFDA)
  43. 51. Surgical procedure
  44. 52. <ul><li>axial stress to the disc : 5 times the animal’s body weight, equivalent to 2.3 MPa, for two weeks </li></ul>
  45. 53. transplantation of 0.08 ml bMSCs solution are injected into disc by posterolateral approach and image intensifier guided through a 25-gauge syringe.
  46. 54. Image intensifier-guided
  47. 55. <ul><li>Cell was additionally analyzed of viable/positive cells from the fresh disc specimen by cell labeling of bMSCs with carboxyfluorescein diacetate (CFDA) to know live cells are come from bMSCs transplantation. </li></ul>Cell labeling
  48. 56. In vivo Cell labeling : 8 weeks after transplantation group 1 (bMSCs) group 4 (distraction and bMSCs)
  50. 58. <ul><li>Better disc height and higher proteoglycan content will be obtained if bMSCs transplantation is combined with intervertebral disc distraction, like what are shown in group 4. </li></ul>
  51. 59. <ul><li>In the group 4 (bMSCs and distraction) : </li></ul><ul><ul><li>the disc height ↑ 16.65% on lateral view and 10.84% on frontal view </li></ul></ul>
  52. 60. Group 3 Group 1 Group 2 Group 4
  53. 61. <ul><li>In the group 4 (bMSCs and distraction) : </li></ul><ul><ul><li>p roteoglycan grade attained 75% on the fourth grade and 25% on the fifth grade. </li></ul></ul>
  54. 62. <ul><li>Axial distraction of the IVD will not only ↑ hidration but also increase disc nutrition. * </li></ul><ul><li>Disc rehidration will ↑ hidrostatic pressure that subsequently will stimulate matrix gene expression, and produce increased extra cellular matrix protein expression . ** </li></ul>* Kroeber M, Unglaub F, Guehring T, Nerlich A, Hadi T, Lotz J, et al. Spine 2005; 2:181-7. * * Guehring T, Omlor GW, Lorenz H, Engelleiter K, Richter W, Kroeber M. Spine 2006;15:1658-65.
  55. 63. <ul><li>The group 4 also showed the best histological score among all groups. </li></ul><ul><li>bMSCs transplantation followed by an increase in disc rehidration gave a good result on cells and matrix extra cellular repair. </li></ul>
  56. 64. <ul><li>The group 2 (distraction of the IVD) : </li></ul><ul><ul><li>least dead cells count (73.35 + 21.55) and significantly difference with control group in all parameters. </li></ul></ul><ul><li>Distraction of the IVD : </li></ul><ul><li>↓ matrix-degradation enzyme, such as MMP-13, which can cause apoptosis pathways to decrease. * </li></ul>* Guehring T et al . Spine 2006; 15 : 1658–1665 Group 3 Group 2
  57. 65. Clinical Implication and Further Study Aspect of bMSCs Aspect of IVD Distraction <ul><li>Regeneration </li></ul><ul><li>Fusion </li></ul><ul><li>In vitro </li></ul><ul><li>In vivo </li></ul><ul><li>Conservative </li></ul><ul><li>Operative </li></ul><ul><li>In vitro </li></ul><ul><li>In vivo </li></ul>
  59. 68. bMSCs transplantation distraction of the IVD to reverse intervertebral disc degeneration in the rabbit or bMSCs transplantation and distraction of the IVD <ul><ul><li>↑ disc height on lateral and frontal view </li></ul></ul><ul><ul><li>Good and statistically significance in </li></ul></ul><ul><ul><li>histological score, morphological and </li></ul></ul><ul><ul><li>proteoglycan grade (except bMSCs </li></ul></ul><ul><ul><li>group in morphological grade) </li></ul></ul><ul><ul><li>↓ the dead cell count </li></ul></ul>↑ restoration IDD toward regeneration in term of lateral projection disc height increase and histological score, although not statistically significant difference compared to bMSCs transplantation only or IVD distraction
  60. 69. Cell Therapy and Stem cells for Cartilage Defect Courtesy of Dr. Andri Lubis, Sp OT Orthopaedic and Traumatology Division Faculty of Medicine University of Indonesia
  61. 71. cartilage MFC denuded 3x5 cm Osteochondral lesion
  62. 73. JBJS Am 2006;88:2502-20 Autologous Chondrocyte Implantation (ACI)
  63. 74. JBJS Am 2006;88:2502-20
  64. 75. ACI Technology Brittberg et al., NEJM 1994; 331:889-95
  65. 76. Matrix-carried Autologous Chondrocyte Implantation (MACI) <ul><li>Cultivated autologous chondrocytes seeded in a 3-D scaffold made of type I/III collagen membrane </li></ul><ul><li>Matrix is remodelled within a few months and replaced by the extracellular matrix of the regenerate </li></ul>
  66. 77. Key Elements of MACI <ul><li>Autologous Chondrocytes: controlled cell density  10M </li></ul><ul><li>Collagen membrane: bio-degradable >6M </li></ul><ul><li>Fibrin glue: promote cell migration and growth (Zheng et al., 2005) </li></ul><ul><li>Histology of MACI: objective evidence (Zheng et al., 2006) </li></ul><ul><li>Functional Assessment: achievement >80% of good to excellent (Robinson et al., 2007) </li></ul>
  67. 78. 3 rd Generation ACI <ul><li>Do not need periosteal patch </li></ul><ul><li>Example: Fibrin Matrix ACI </li></ul>
  68. 79. J Kor Arthros Soc 2007;11:1-5
  69. 80. Bone Marrow-Derived Mesenchymal Stem Cells
  70. 81. BMSCs Steps <ul><li>Taking the stem cell from illiac crest </li></ul><ul><li>Culture the stem cells  4 weeks lab </li></ul><ul><li>Debridement the lession </li></ul><ul><li>Taking tibial periosteum </li></ul><ul><li>Suturing the periost to the defect, implantation of stem cells, completing the suture </li></ul><ul><li>Fibrin glue </li></ul>
  71. 82. Courtesy of Prof. James Hui, National University Hospital, Singapore
  72. 85. Courtesy of Prof. James Hui, NUH
  73. 86. Radiographics 2007; 27:207-22
  74. 87. Eur Radiol 2007; 17:103–18
  75. 88. The success of Stem Cells Therapy <ul><li>Need good collaboration among surgeons, laboratory technicians, and stem cells laboratory </li></ul>Dr. Teo Cheng Peng Parkway Cancer Center, S’pore Kompas March 16, 2007 p. 42
  76. 89. Tissue Engineering and Cell Based Therapies: a 21 st Century Treatment Dr. Caplan
  77. 91. The End Thank you for the attention