||Osteochondral Regeneration using PLGA Scaffolds under Short-term Continuous Passive Motion in a Rabbit Model
||Osteochondral Regeneration using PLGA Scaffolds under Short-term Continuous Passive Motion in a Rabbit Model
||Institute of Biomedical Engineering
continuous passive motion
cartilage tissue engineering
continuous passive motion
cartilage tissue engineering
在軟骨組織工程中，細胞、細胞支架和刺激是三個重要的組成。骨軟骨病是一種軟骨內骨化在生長板上的病變，因而導致骨關節炎或退化性軟骨疾病。連續式被動運動是物理性刺激，它具有刺激細胞在關節缺陷處之增生。為了更加了解連續式被動運動如何在全層軟骨缺陷生成修復之效應，本研究使用兔子模型經由評估巨觀、電腦斷層掃描以及組織學染色。三釐米大小的缺陷分別在髕骨溝和內側髁各代表高承重區和低承重區。聚乳酸甘醇酸是生醫可降解高分子，它已經被美國食品及藥物管理局所認可使用且廣泛地應用於生醫材料產品中。在本研究中，聚乳酸甘醇酸經由鹽析法製備圓柱型多孔洞細胞支架。細胞支架被植入於骨軟骨缺陷之實驗組。每組三隻兔子治療於每一組,觀察時間點為:有/無聚乳酸甘醇酸結合固定膝關節在術後四週；有/無聚乳酸甘醇酸結合固定膝關節在術後十二週；有/無聚乳酸甘醇酸結合自由活動在術後四週；有/無聚乳酸甘醇酸結合自由活動在術後十二週; 有/無聚乳酸甘醇酸結合連續式被動運動在術後四週; 有/無聚乳酸甘醇酸結合連續式被動運動在術後十二週。結果顯示在術後四週，聚乳酸甘醇酸細胞支架提供力學性質在內側髁。雖然組織學分數在固定膝關節結合聚乳酸甘醇酸細胞支架組最高，但異常骨頭增生被發現且限制了關節角度。術後十二週，細胞支架的修復在內側髁經由連續式被動運動結合細胞支架組顯著性高於只有連續式被動運動(p = 0.048)。總結，關節軟骨之功能和細胞分化與增生在修復成透明軟骨扮演了重要角色。本研究顯示連續式被動運動結合細胞支架組可以誘發軟骨和硬骨分化在修復處，此方法也許可應用於臨床和軟骨組織工程。
Cells, scaffolds, and stimulation are three important components in cartilage tissue engineering. Osteochondrosis is a disorder of endochondral ossification in epiphyseal growth plate, leading to the osteoarthritis or the degenerative joint cartilage. Continuous passive motion (CPM), a natural physiological stimulation, should promote cell proliferation in joint defect regeneration. To better understand how continuous passive motion affects on the formation of repairing tissue in full-thickness cartilage defects, this study evaluated the effects of CPM in articular defects in rabbit model by macroscopic observation, computed tomography scanning, and histological evaluation. A 3-mm depth from the articular surface was produced in the patella groove and the medial condyle respectively to represent the high- and low- weight-bearing regions. Poly DL-lactic-co-glycolic acid (PLGA) that is a biodegradable polymer approved by FDA is widely used in biomaterial products. PLGA scaffolds were fabricated into the cylinder porous sponges by salt-leaching technique. The scaffolds were placed into the osteochondral defects for experimental group. Thirty-six rabbits were separated into six groups which were treated in individual study: with/without PLGA scaffold with immobilization on week 4 and 12; with/without PLGA scaffold with free cage activity for 4 and 12 weeks; and with/without PLGAscaffold with CPM on week 4 and 12. The results at four weeks showed the use of PLGA scaffold provided the mechanical functions at the medial condyle. Even though the histological scores of the immobilization treated with PLGA scaffold showed the highest, there was osteophyte which can limit the range of motion on the joint. After 12 weeks post-operation, the healing process of scaffold treated CPM were significantly better than that CPM alone at the medial condyle (p = 0.048). In conclusion, the articular cartilage function, and cell differentiation and proliferation are important to regenerate hyaline cartilage. This study showed that the scaffolds in combination of CPM can induce both chondrogenesis and osteogenesis in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.
TABLE OF CONTENTS II
LIST OF TABLES IV
LIST OF FIGURES V
CHAPTER ONE INTRODUCTION 1
1.1 Background. 1
1.2 Structure of articular cartilage. 2
1.3 Osteochondral repair and regenerative techniques. 5
1.4 Chondrogenesis and chondrocyte differentiation. 6
1.5 Cartilage tissue engineering. 7
1.5.1 Scaffold properties. 8
1.5.2 Stimulation – CPM. 8
1.4 Literature review. 9
1.5 Previous study 12
1.6 Motivation. 13
1.6 Purpose. 13
CHAPTER TWO MATERIALS AND METHODS 15
2.1 Flow Chart. 15
2.2 Materials 16
2.3 Preparation and mechanical properties of PLGA scaffolds. 17
2.4 Experiment procedures. 18
2.5 Macroscopic evaluation. 21
2.6 Micro-CT scanning. 21
2.7 Histological techniques and scoring system. 22
2.7 Statistics. 25
CHAPTER THREE RESULTS 26
3.1 Rabbit circumstances. 26
3.2 Gross appearance. 27
3.3 Bone formation analysis by micro-CT 31
3.4 Histological examination. 34
CHAPTER FOUR DISCUSSION 43
4.1 PLGA implantation. 43
4.2 Animal study. 43
4.3 Time period. 44
4.4 Cells and histological observation. 45
4.5 Limitations. 46
4.8 Future work. 48
CHAPTER 5 CONCLUSION 49
1. Convery, F.R., G.H. Keown, and W.H. Akeson, Repair of Large Osteochondral Defects - Experimental Study in Horses. Clinical Orthopaedics and Related Research, 1972(82): p. 253-&.
2. Shapiro, F., S. Koide, and M.J. Glimcher, Cell Origin and Differentiation in the Repair of Full-Thickness Defects of Articular-Cartilage. Journal of Bone and Joint Surgery-American Volume, 1993. 75A(4): p. 532-553.
3. Brittberg, M., et al., Rabbit Articular Cartilage Defects Treated With Autologous Cultured Chondrocytes. Clinical Orthopaedics and Related Research, 1996. 326: p. 270-283.
4. O'Driscoll, S.W., The healing and regeneration of articular cartilage. J Bone Joint Surg Am, 1998. 80(12): p. 1795-812.
5. Salter, R.B., et al., The Biological Effect of Continuous Passive Motion on the Healing of Full-Thickness Defects in Articular-Cartilage - an Experimental Investigation in the Rabbit. Journal of Bone and Joint Surgery-American Volume, 1980. 62(8): p. 1232-1251.
6. Vanroyen, B.J., et al., A Comparison of the Effects of Immobilization and Continuous Passive Motion on Surgical Wound-Healing in Mature Rabbits. Plastic and Reconstructive Surgery, 1986. 78(3): p. 360-366.
7. Ferretti, M., et al., Anti-inflammatory effects of continuous passive motion on meniscal fibrocartilage. Journal of Orthopaedic Research, 2005. 23(5): p. 1165-1171.
8. Kim, H.K.W., et al., The effects of postoperative continuous passive motion on peripheral nerve repair and regeneration - An experimental investigation in rabbits. Journal of Hand Surgery-British and European Volume, 1998. 23B(5): p. 594-597.
9. Newman, A.P., Articular cartilage repair. American Journal of Sports Medicine, 1998. 26(2): p. 309-324.
10. Hunziker, E.B., Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis and Cartilage, 2002. 10(6): p. 432-463.
11. O'Driscoll, S.W., F.W. Keeley, and R.B. Salter, The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the influence of continuous passive motion. An experimental investigation in the rabbit. J Bone Joint Surg Am, 1986. 68(7): p. 1017-35.
12. Furukawa, T., et al., Biochemical-Studies on Repair Cartilage Resurfacing Experimental Defects in the Rabbit Knee. Journal of Bone and Joint Surgery-American Volume, 1980. 62(1): p. 79-89.
13. Shao, X.X., et al., Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits. Biomaterials, 2006. 27(7): p. 1071-1080.
14. Wakitani, S., et al., Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am, 1994. 76(4): p. 579-92.
15. Kang, H.J., et al., An experimental intraarticular implantation of woven carbon fiber pad into osteochondral defect of the femoral condyle in rabbit. Yonsei Med J, 1991. 32(2): p. 108-16.
16. Zhang, L. and M. Spector, Tissue Engineering of Musculoskeletal Tissue, in Tissue Engineering, N. Pallua and C.V. Suscheck, Editors. 2011, Springer Berlin Heidelberg. p. 597-624.
17. Angermann, P., K. Harager, and L.L. Tobin, Arthroscopic chondrectomy as a treatment of cartilage lesions. Knee Surgery Sports Traumatology Arthroscopy, 2002. 10(1): p. 6-9.
18. Bae, D.K., K.H. Yoon, and S.J. Song, Cartilage Healing After Microfracture in Osteoarthritic Knees. Arthroscopy: The Journal of Arthroscopic & Related Surgery, 2006. 22(4): p. 367-374.
19. Minas, T. and L. Peterson, Advanced techniques in autologous chondrocyte transplantation. Clinics in Sports Medicine, 1999. 18(1): p. 13-+.
20. Zuscik, M.J., et al., Regulation of chondrogenesis and chondrocyte differentiation by stress. Journal of Clinical Investigation, 2008. 118(2): p. 429-438.
21. Wagner, W., et al., Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Experimental Hematology, 2005. 33(11): p. 1402-1416.
22. Langer, R. and J.P. Vacanti, Tissue Engineering. Science, 1993. 260(5110): p. 920-926.
23. Chaubal, M., Polylactides/glycolides-excipients for injectable drug delivery and beyond. Drug Delivery Technology, 2002. 2(5): p. 34,36.
24. Lan, P. and L. Jia, Thermal properties of copoly(L-lactic acid/glycolic acid) by direct melt polycondensation. Journal of Macromolecular Science Part a-Pure and Applied Chemistry, 2006. 43(11): p. 1887-1894.
25. Chiu Lin, P. 510(k) Summary in accordance with 21 CFR 807.92(c). 2008; Available from: http://www.accessdata.fda.gov/cdrh_docs/pdf8/K080308.pdf.
26. O'Driscoll, S.W. and N.J. Giori, Continuous passive motion (CPM): Theory and principles of clinical application. Journal of Rehabilitation Research and Development, 2000. 37(2): p. 179-188.
27. Lenssen, T.A.F., et al., Effectiveness of prolonged use of continuous passive motion (CPM), as an adjunct to physiotherapy, after total knee arthroplasty. Bmc Musculoskeletal Disorders, 2008. 9: p. -.
28. Richmond, J.C., J. Gladstone, and J. MacGillivray, Continuous passive motion after arthroscopically assisted anterior cruciate ligament reconstruction: Comparison of short- versus long-term use. Arthroscopy: The Journal of Arthroscopic & Related Surgery, 1991. 7(1): p. 39-44.
29. Nishino, T., et al., Effect of Gradual Weight-Bearing on Regenerated Articular Cartilage after Joint Distraction and Motion in a Rabbit Model. Journal of Orthopaedic Research, 2010. 28(5): p. 600-606.
30. Howard, J.S., et al., Continuous Passive Motion, Early Weight Bearing, and Active Motion following Knee Articular Cartilage Repair. Cartilage, 2010. 1(4): p. 276-286.
31. Mandelbaum, B.R., et al., Articular cartilage lesions of the knee. American Journal of Sports Medicine, 1998. 26(6): p. 853-861.
32. A. Getgood, T.P.S.B., and N. Rushton, Current concepts in articular cartilage repair. Orthopaedics and Trauma, 2009. 23(3): p. 189-200.
33. Steadman, J.R., et al., Microfracture technique forfull-thickness chondral defects: Technique and clinical results. Operative Techniques in Orthopaedics, 1997. 7(4): p. 300-304.
34. Hutmacher, D.W. and A.J. Garcia, Scaffold-based bone engineering by using genetically modified cells. Gene, 2005. 347(1): p. 1-10.
35. Chung, C. and J.A. Burdick, Engineering cartilage tissue. Adv Drug Deliv Rev, 2008. 60(2): p. 243-62.
36. Buckley, C.T. and K.U. O'Kelly, Maintaining cell depth viability: on the efficacy of a trimodal scaffold pore architecture and dynamic rotational culturing. Journal of Materials Science-Materials in Medicine, 2010. 21(5): p. 1731-1738.
37. Swieszkowski, W., et al., Repair and regeneration of osteochondral defects in the articular joints. Biomol Eng, 2007. 24(5): p. 489-95.
38. Woodfield, T.B.F., et al., Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique. Biomaterials, 2004. 25(18): p. 4149-4161.
39. Uematsu, K., et al., Cartilage regeneration using mesenchymal stem cells and a three-dimensional poly-lactic-glycolic acid (PLGA) scaffold. Biomaterials, 2005. 26(20): p. 4273-9.
40. Wu, W.G., et al., A programmed release multi-drug implant fabricated by three-dimensional printing technology for bone tuberculosis therapy. Biomedical Materials, 2009. 4(6): p. -.
41. Vozzi, G., et al., Microfabricated PLGA scaffolds: a comparative study for application to tissue engineering. Materials Science & Engineering C-Biomimetic and Supramolecular Systems, 2002. 20(1-2): p. 43-47.
42. Yang, R., et al., Microfabrication of biodegradable (PLGA) honeycomb-structures and potential applications in implantable drug delivery. Sensors and Actuators B-Chemical, 2005. 106(2): p. 506-511.
43. Lin, H.R., et al., Preparation of macroporous biodegradable PLGA scaffolds for cell attachment with the use of mixed salts as porogen additives. Journal of Biomedical Materials Research, 2002. 63(3): p. 271-279.
44. Leung, L., et al., Comparison of morphology and mechanical properties of PLGA bioscaffolds. Biomedical Materials, 2008. 3(2): p. -.
45. Lu, L., et al., In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams. Biomaterials, 2000. 21(18): p. 1837-1845.
46. Han, S.H., et al., Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo. Journal of Biomedical Materials Research Part A, 2008. 87A(4): p. 850-861.
47. Cheuk, Y.-C., et al., Use of allogeneic scaffold-free chondrocyte pellet in repair of osteochondral defect in a rabbit model. Journal of Orthopaedic Research, 2011. 29(9): p. 1343-1350.
48. Kumagai, K., T. Saito, and T. Koshino, Articular cartilage repair of rabbit chondral defect: promoted by creation of periarticular bony defect. Journal of Orthopaedic Science, 2003. 8(5): p. 700-706.
49. Han, S.H., et al., Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo. Journal of Biomedical Materials Research Part A, 2008. 87A(4): p. 850-861.
50. Chu, C.R., M. Szczodry, and S. Bruno, Animal Models for Cartilage Regeneration and Repair. Tissue Engineering Part B-Reviews, 2010. 16(1): p. 105-115.
51. Salter, R.B., et al., The biological effect of continuous passive motion on the healing of full-thickness defects in articular cartilage. An experimental investigation in the rabbit. J Bone Joint Surg Am, 1980. 62(8): p. 1232-51.
52. Nugent-Derfus, G.E., et al., Continuous passive motion applied to whole joints stimulates chondrocyte biosynthesis of PRG4. Osteoarthritis Cartilage, 2007. 15(5): p. 566-74.
53. Gebhard, J.S., J.M. Kabo, and R.A. Meals, Passive Motion - the Dose Effects on Joint Stiffness, Muscle Mass, Bone-Density, and Regional Swelling. Journal of Bone and Joint Surgery-American Volume, 1993. 75A(11): p. 1636-1647.
54. Shimizu, T., et al., Experimental-Study on the Repair of Full Thickness Articular-Cartilage Defects - Effects of Varying Periods of Continuous Passive Motion, Cage Activity, and Immobilization. Journal of Orthopaedic Research, 1987. 5(2): p. 187-197.
55. O'Driscoll, S.W., F.W. Keeley, and R.B. Salter, Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. A follow-up report at one year. J Bone Joint Surg Am, 1988. 70(4): p. 595-606.
56. Frenkel, S.R., et al., Regeneration of articular cartilage--evaluation of osteochondral defect repair in the rabbit using multiphasic implants. Osteoarthritis Cartilage, 2005. 13(9): p. 798-807.
57. Mainil-Varlet, P., et al., A New Histology Scoring System for the Assessment of the Quality of Human Cartilage Repair: ICRS II. American Journal of Sports Medicine, 2010. 38(5): p. 880-890.
58. Martin-Hernandez, C., et al., Regenerated Cartilage Produced by Autogenous Periosteal Grafts: A Histologic and Mechanical Study in Rabbits Under the Influence of Continuous Passive Motion. Arthroscopy-the Journal of Arthroscopic and Related Surgery, 2010. 26(1): p. 76-83.
59. Wu, L.B., et al., "Wet-state" mechanical properties of three-dimensional polyester porous scaffolds. Journal of Biomedical Materials Research Part A, 2006. 76A(2): p. 264-271.
60. Jhung, Y.-R., The Application of Hydrodynamic Pressure Bioreactor on Chondrocytes Cultured on 3-D Hydrophilic PLGA Scaffold, in Institute of Biomedical Engineering. 2010, National Cheng Kung University: Tainan.
61. Wayne, J.S., et al., In vivo response of polylactic acid-alginate scaffolds and bone marrow-derived cells for cartilage tissue engineering. Tissue Engineering, 2005. 11(5-6): p. 953-963.
62. Song, S.U., et al., Hyaline cartilage regeneration using mixed human chondrocytes and transforming growth factor-beta(1)-producing chondrocytes. Tissue Engineering, 2005. 11(9-10): p. 1516-1526.
63. Holland, T.A., et al., Degradable hydrogel scaffolds for in vivo delivery of single and dual growth factors in cartilage repair. Osteoarthritis and Cartilage, 2007. 15(2): p. 187-197.
64. Ouyang, H.W., et al., The Inductive Effect of Bone Morphogenetic Protein-4 on Chondral-Lineage Differentiation and In Situ Cartilage Repair. Tissue Engineering Part A, 2010. 16(5): p. 1621-1632.
65. Guoping Chen, T.S., Junzo Tanaka and Tetsuya Tateishi, Preparation of a biphasic scaffold for osteochondral tissue engineering. Materials Science and Engineering: C, 2005. 26(1): p. 118-123.
66. Kavanagh, E. and D.E. Ashhurst, Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1. Journal of Histochemistry & Cytochemistry, 1999. 47(12): p. 1603-1615.
67. Hurtig, M.B., et al., Preclinical Studies for Cartilage Repair. Cartilage, 2011. 2(2): p. 137-152.
68. Mano, J.F. and R.L. Reis, Osteochondral defects: present situation and tissue engineering approaches. Journal of Tissue Engineering and Regenerative Medicine, 2007. 1(4): p. 261-273.
69. Yoshimura, N., et al., Association of Knee Osteoarthritis, Lumbar Spondylosis and Osteoporosis with Metabolic Syndrome: The Road Study. Osteoporosis International, 2010. 21: p. 286-286.
70. Videman, T., Changes in compression and distances between tibial and femoral condyles during immobilization of rabbit knee. Archives of Orthopaedic and Trauma Surgery, 1981. 98(4): p. 289-291.
71. Siddiqui, M.A. and M.H. Tan, Locked knee from superior dislocation of the patella-diagnosis and management of a rare injury. Knee Surgery Sports Traumatology Arthroscopy, 2011. 19(4): p. 671-673.
72. Xu, Y. and Y.F. Ao, Histological and biomechanical studies of inter-strand healing in four-strand autograft anterior cruciate ligament reconstruction in a rabbit model. Knee Surgery Sports Traumatology Arthroscopy, 2009. 17(7): p. 770-777.
73. Mullender, M.G. and R. Huiskes, Osteocytes and bone lining cells: Which are the best candidates for mechano-sensors in cancellous bone? Bone, 1997. 20(6): p. 527-532.
74. Jacobs, C.R., S. Temiyasathit, and A.B. Castillo, Osteocyte Mechanobiology and Pericellular Mechanics. Annual Review of Biomedical Engineering, Vol 12, 2010. 12: p. 369-400.
75. Olivos-Meza, A., et al., Pretreatment of periosteum with TGF-[beta]1 in situ enhances the quality of osteochondral tissue regenerated from transplanted periosteal grafts in adult rabbits. Osteoarthritis and Cartilage, 2010. 18(9): p. 1183-1191.
76. Rudert, M., Histological evaluation of osteochondral defects: Consideration of animal models with emphasis on the rabbit, experimental setup, follow-up and applied methods. Cells Tissues Organs, 2002. 171(4): p. 229-240.
77. Safety and Complications Reporting on the Re-implantation of Culture-Expanded Mesenchymal Stem Cells using Autologous Platelet Lysate Technique. Current Stem Cell Research & Therapy, 2010. 5: p. 81-93.
78. Minas, T., et al., Autologous Chondrocyte Implantation for Joint Preservation in Patients with Early Osteoarthritis. Clinical Orthopaedics and Related Research®, 2010. 468(1): p. 147-157.
79. Basad, E., et al., Matrix-induced autologous chondrocyte implantation versus microfracture in the treatment of cartilage defects of the knee: a 2-year randomised study. Knee Surgery, Sports Traumatology, Arthroscopy, 2010. 18(4): p. 519-527.
80. Miller, R.E., et al., Effect of self-assembling peptide, chondrogenic factors, and bone marrow-derived stromal cells on osteochondral repair. Osteoarthritis and Cartilage, 2010. 18(12): p. 1608-1619.