臺灣博碩士論文加值系統

中文摘要
骨質疏鬆(Osteoporosis)是殘障病人常見併發症，這些骨質疏鬆的病人，常因日常生活中輕微受傷就造成骨折。骨折後之癒合也是極度複雜的過程，癒合不良在臨床上往往造成高致病率。因此，如何預防骨質疏鬆與避免骨折，以及骨折後如何防止癒合不良，是很重要的課題。細胞接受機械拉力後，會造成骨骼系統之生化反應而刺激骨生成，超音波(non-invasive low-energy ultrasound)及功能性電刺激踩車系統 (functional electrical stimulation cycling exercises) 在骨骼系統會有類似機械拉力的作用，因此本博士論文分成二部份：包括超音波對於人類間葉幹細胞分化成軟骨與骨細胞的影響之實驗及功能性電刺激踩車在骨質流失之臨床研究。
第一部份之研究目的為：探討超音波及細胞激素對於人類間葉幹細胞(human mesenchymal stem cells ;hMSCs) 軟骨分化(chondrogenic differentiation)與骨分化(osteogenic differentiation)之影響。此研究分別用合成皮質類固醇 / 轉化生長因子(dexamethasone/transforming growth factor :TD)或骨頭生成蛋白質(bone morphogenetic protein-2 ;BMP-2)下，在有或無給予超音波處置下，人類間葉幹細胞軟骨化或骨化之變化。我們分析細胞外型變化，並運用及時聚合鏈反應(real-time polymerase chain reaction )分析軟骨化(chondrogenic)及骨化(osteogenic)之基因表現 (messenger RNA expression)。TD處置之人類間葉幹細胞顯示明顯軟骨化細胞形狀及增加軟骨化基因表現，給予超音波和TD處置預期有加乘作用。就骨化的基因表現而言，超音波和骨生成蛋白質分別都會刺激骨化之基因表現，但二者一起作用卻沒有顯示明顯加乘效果。因此超音波可能對於間葉細胞軟骨分化及骨分化有不同影響。這個結果可以提供在使用超音波促進軟骨及骨頭修復上之臨床參考。
第二部分之研究為與骨密度(bone mineral density)相關之臨床實驗，這個研究主要目的為早期給予脊髓損傷者(spinal cord injury)功能性電刺激踩車訓練，是否可以預防或減輕骨質疏鬆？另外也分析若停止踩車訓練後，此一預防或減輕骨質疏鬆的效果是否依然存在？本研究共有24位受傷後26至52天之脊髓損傷患者參加，其中12位加入功能性電刺激踩車組，另外12位為控制組。功能性電刺激踩車組患者接受3個月踩車訓練，然後停止訓練3個月，患者之股骨頸(femoral neck )及遠端股骨(distal femur)分別於訓練前後三個月後，及停止訓練三個月後，運用雙光光子測量法(dual energy X-ray absorptiometry)來測量骨密度，結果顯示：在功能性電刺激踩車訓練三個月期間，訓練組之遠端股骨骨密度下降率明顯少於控制組，但是停止訓練三個月期間，二組間遠端股骨骨密度下降率並無差異。因此，功能性電刺激踩車訓練可以部份減輕遠端股骨之骨密度流失，但是此訓練無法完全阻止骨密度流失。另外，在停止訓練後，減輕骨密度流失之效果無法持續。
由上述二部份研究讓我們了解到，超音波和功能性電刺激踩車這二種會產生機械拉力的物理因子，會對骨骼系統產生正向影響。超音波會改善間葉幹細胞往軟骨及骨細胞分化，而功能性電刺激踩車訓練可以減輕遠端股骨骨質的流失。

Abstract
Osteoporosis is a well-recognized complication among disabled individuals. Secondary to the characteristic bone loss of osteoporosis, fractures caused by minimal trauma often occur among these individuals. Therefore, prevention of osteoporosis and subsequent reduction in fractures due to bone loss in disabled subjects is an important issue. Additionally, healing of fractures is an extremely complex process; delayed union or non-union are frequently observed in clinical practice and present potential morbidities. The mechanical strain received by cells may result in biochemical events that, in the skeleton, have been shown to promote bone formation. Non-invasive low intensity pulsed ultrasound (LIPUS) and functional electrical stimulation cycling exercises (FESCE) can mimic the effects of mechanical loading on bone. This dissertation addresses these treatment modalities and consists of two parts: the investigation of LIPUS in in vitro models and the investigation of FESCE in clinical circumstances involving bone regeneration.
In the first part of this study, the effects of LIPUS on the differentiation of human mesenchymal stem cells (hMSCs) were investigated. The hMSCs were subjected to LIPUS either with or without dexamethasone/transforming growth factor-β1 (TD) or bone morphogenetic protein-2 (BMP-2), and the effects of these treatments were assessed in two groups, a LIPUS-only group and a LIPUS-with-TD treatment group. The TD-treated hMSCs exhibited characteristic chondrogenic morphology and increased mRNA expression of chondrogenic markers, and LIPUS enhanced the chondrogenic differentiation of hMSCs treated with TD. No alterations were seen in the expression of Runx2, an osteogenic transcription factor, in either the LIPUS-only group or the LIPUS-with-TD treatment group; however, a significant increase was detected in the LIPUS-only group. The osteogenic appearance was seen three days after LIPUS and/or BMP-2 treatment. Increases in the mRNA expression levels of osteogenic markers, Runx2 and ALP were also detected. No additive or altered effects were noted with combined LIPUS and BMP-2 treatment. LIPUS alone can increase osteogenic differentiation of hMSCs and LIPUS enhances TD-mediated chondrogenic differentiation of hMSCs. Clinically, LIPUS may differentially influence bone versus cartilage repair.
The second part of this study was designed to determine whether loss of bone mineral density (BMD) after spinal cord injury (SCI) can be attenuated by early intervention with FESCE and to ascertain whether the effect persists after FESCE is discontinued.
Twenty-four patients with SCI, 26-52 days after injury, were divided into a FESCE group and a control group. In the treatment group, FESCE was applied in the initial three months and then suspended in the subsequent three months. BMDs of the femoral neck and distal femur (DF) were obtained by dual energy X-ray absorptiometry (DXA) before training, immediately after the initial three months of training, and at the end of the subsequent three months. During the initial three months, the reduction rate for BMD in the DF of the FESCE group was significantly less than that of the control group. However, no significant differences were noted in the subsequent three months when treatment was suspended. In summary, FESCE in the early stages of SCI can partly attenuate BMD loss in the DF. However, BMD loss in the DF cannot be ameliorated completely by FESCE. In addition, the effect on the attenuation of bone loss in the DF faded once FESCE was discontinued.
These findings suggest that LIPUS and FESCE mimicking mechanical strain may have a positive skeletal impact. LIPUS enhances chondrogenic differentiation of hMSCs that have received TD induction and promotes osteogenic differentiation of hMSC. FESCE applied in the early stages of SCI can partly attenuate BMD loss in the DF.

Contents
中文摘要……………………………………………………………………….I
Abstract…………………………………………………………......................IV
Acknowledgement…………………………………………………………….VII
Contents………………………………………………………………………..VIII
List of Figures…………………………………………………………………..XI
List of Tables……………………………………………………………………XIII
Scope of the Thesis……………………….…………………………..................1
Part I Regulation of Chondrogenic and Osteogenic Differentiation of Human
Mesenchymal Stem Cells Using Low-Intensity Pulsed Ultrasound (LIPUS), Dexamethasone/ TGF-β1, and BMP-2………………………………….9
Chapter 1 Introduction of the effects LIPUS and cytokines on chodrogenic and
osteogenic differentiation of hMSCs…………………………………….10
Chapter 2 Materials and Methods of hMSCs differentiation and LIPUS
application………………………………………………………….....13
2.1 Subjects……………………………………………………………………...13
2.2 Isolation and cultivation of hMSCs………………………………………….13
2.3 Analysis of chondrogenic differentiation…………………………………....14
2.4 Analysis of osteogenic differentiation……………………………………….15
2.5 Application of LIPUS………………………………………………………..15
2.6 Real-time PCR…………………………………………………….................22
2.7 Statistical analysis………………………………………………………..23
Chapter 3 Results of morphological changes and transcriptional expressions on chodrogenic and osteogenic differentiation of hMSCs………………...24
3.1 Morphological changes in the chondrogenic differentiation of hMSCs…..24
3.2 Real-time PCR results of the chondrogenic differentiation of hMSCs……27
3.3 Morphological changes in the osteogeic differentiation of hMSCs……… 36
3.4 Real-time PCR results of the osteogenic differentiation of hMSCs………37
Chapter 4 Discussion of the effects of LIPUS and cytokines on chodrogenic and
osteogenic differentiation of hMSCs……………………………………42
Chapter 5 Conclusion of the effects of LIPUS and cytokines on chodrogenic and
osteogenic differentiation of hMSCs …………………………………..48
Part II Effects of Functional Electrical Stimulation Cycling Exercise on Bone
Mineral Density Loss in the Early Stage of Spinal Cord Injury…………49
Chapter 6 Introduction of the effects of FESCE on BMD loss in individuals with
SCI……………………………………………………………………50
Chapter 7 Materials and Methods of FESCE programs and BMD measurements..53
7.1 Individuals…………………………………………………………………53
7.2 FESCE Program…………………………………………………………...55
7.3 BMD Measurements…..……………………………………………………58
7.4 Data analysis…..………………………………………………………….59
Chapter 8 Results of BMD loss in the individuals with SCI ………………......60
Chapter 9 Discussion of the effects of FESCE on BMD loss in the early stage of SCI …………………………………………………………………..67
Chapter 10 Conclusion of the effects of FESCE on BMD loss in the early stage of
SCI……………………………………………………………………71
Chapter 11 Summary and Prospects……………………………………………..72
References …………………………………………………………………………74
Appendix …. ………………………………………………………………………98

List of Figures
Fig.1 (A) Diagram of the experimental setup for exposure of mesenchymal stem cells
(MSCs) grown as a monolayer to low intensity pulse ultrasound (LIPUS). (B)
An enlarged diagram of culture dish (CD) covered by specific absorption
chamber (SAC) of the LIPUS exposure system…………………………….18
Fig.2 Measurement of axial attenuation of the transducer transmission of acoustic
waves (1 MHz, 200 mW) through distilled water……………………………..20
Fig.3 Morphological changes in the chondrogenic differentiation of human
mesenchymal stem cells (hMSCs)……………………..……………………...26
Fig.4 Box plots of mRNA expression levels of integrin β1, Sox9, aggrecan,
type I collagen, type II collagen, and Runx2 under different treatment
conditions……………………………………………………………………...32
Fig.5 Morphological changes in the osteogenic differentiation of human
mesenchymal stem cells (hMSCs)…………………………………………....36
Fig.6 Effect of low intensity pulse ultrasound (LIPUS) and/or bone morphogenetic protein (BMP) treatment on expression of genes related to osteogenic differentiation of human mesenchymal stem cells (hMSC)…………………...39
Fig.7 Protocol of functional electrical stimulation cycling exercise (FESCE) training………………………………………………………………………...57
Fig. 8 Normalized bone mineral density (BMD) at the first, second, and third
measurements and the BMD decrease rate in the functional electrical
stimulation cycling exercise (FESCE) and control groups……………………65

List of Tables
Table 1 Summary of the transcriptional expression of integrinβ1, sox 9, aggrecan, type I collagen, and type II collagen in the chonfrogenic differentiation of hMSCs under different treatments………………………………………….28
Table 2 Summary of the transcriptional expression of ALP, Runx2, type I collagen,
and type II collagen in the osteogenic differentiation of hMSCs under
different treatments …………………………………………………………38
Table 3 Characteristics of spinal cord injury subjects in the functional electrical stimulation cycling exercise (FESCE) and control groups…………..............54
Table 4 Bone mineral density (BMD) (mean  SE) and normalized BMD (%) at first,
second, and third measurements in the femoral neck in the functional electrical
stimulation cycling exercise (FESCE) and control groups…………………..61
Table 5 Bone mineral density (BMD) (mean ± SE) and normalized BMD (%) at first,
second, and third measurements in the distal femur in the functional electrical
stimulation cycling exercise (FESCE) and control groups…………………63
Table 6 Comparison of bone mineral density decreases in the functional electrical
stimulation cycling exercise (FESCE) and control groups at each site in
initial and subsequent 3-month periods…………………………………….64

References:
[1]U. A. Liberman, et al., "Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. The Alendronate Phase III Osteoporosis Treatment Study Group," New England Journal of Medicine, vol. 333, pp. 1437-43, Nov 30 1995.
[2]F. Tabrah, et al., "Bone density changes in osteoporosis-prone women exposed to pulsed electromagnetic fields (PEMFs)," Journal of Bone & Mineral Research, vol. 5, pp. 437-42, May 1990.
[3]O. Johnell and J. A. Kanis, "An estimate of the worldwide prevalence and disability associated with osteoporotic fractures," Osteoporosis International, vol. 17, pp. 1726-33, Dec 2006.
[4]C. T. Rubin and L. E. Lanyon, "Regulation of bone mass by mechanical strain magnitude," Calcified Tissue International, vol. 37, pp. 411-7, Jul 1985.
[5]C. H. Turner and F. M. Pavalko, "Mechanotransduction and functional response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation," Journal of Orthopaedic Science, vol. 3, pp. 346-55, 1998.
[6]H. M. Frost, "Skeletal structural adaptations to mechanical usage (SATMU): 2. Redefining Wolff's law: the remodeling problem," Anatomical Record, vol. 226, pp. 414-22, Apr 1990.
[7]H. M. Frost, "Skeletal structural adaptations to mechanical usage (SATMU): 1. Redefining Wolff's law: the bone modeling problem," Anatomical Record, vol. 226, pp. 403-13, Apr 1990.
[8]S. J. Warden, et al., "Skeletal effects of low-intensity pulsed ultrasound on the ovariectomized rodent," Ultrasound in Medicine & Biology, vol. 27, pp. 989-98, Jul 2001.
[9]J. D. Heckman, et al., "Acceleration of tibial fracture-healing by non-invasive, low-intensity pulsed ultrasound.[see comment]," Journal of Bone & Joint Surgery - American Volume, vol. 76, pp. 26-34, Jan 1994.
[10]J. S. Sun, et al., "In vitro effects of low-intensity ultrasound stimulation on the bone cells," Journal of Biomedical Materials Research, vol. 57, pp. 449-56, Dec 5 2001.
[11]F. Ozdemir, et al., "Evaluation of the efficacy of therapeutic ultrasound on bone mineral density in postmenopausal period," Rheumatology International, vol. 28, pp. 361-5, Feb 2008.
[12]S. J. Warden, et al., "Efficacy of low-intensity pulsed ultrasound in the prevention of osteoporosis following spinal cord injury," Bone, vol. 29, pp. 431-6, Nov 2001.
[13]C. L. Tsai, et al., "Ultrasound can affect bone healing both locally and systemically," Chinese Journal of Physiology, vol. 34, pp. 213-22, 1991.
[14]G. R. ter Haar and S. Daniels, "Evidence for ultrasonically induced cavitation in vivo," Physics in Medicine & Biology, vol. 26, pp. 1145-9, Nov 1981.
[15]W. L. Nyborg, "Ultrasonic microstreaming and related phenomena," British Journal of Cancer - Supplement, vol. 5, pp. 156-60, Mar 1982.
[16]Y. Azuma, et al., "Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus," Journal of Bone & Mineral Research, vol. 16, pp. 671-80, Apr 2001.
[17]N. M. Rawool, et al., "Power Doppler assessment of vascular changes during fracture treatment with low-intensity ultrasound," Journal of Ultrasound in Medicine, vol. 22, pp. 145-53, Feb 2003.
[18]A. Frotzler, et al., "High-volume FES-cycling partially reverses bone loss in people with chronic spinal cord injury," Bone, vol. 43, pp. 169-76, Jul 2008.
[19]Y. H. Lee, et al., "The effect of early therapeutic electrical stimulation on bone mineral density in the paralyzed limbs of the rabbit," Yonsei Medical Journal.42(2):194-8, 2001.
[20]R. K. Shields, et al., "Electrically induced muscle contractions influence bone density decline after spinal cord injury," Spine, vol. 31, pp. 548-53, Mar 1 2006.
[21]M. Hartig, et al., "Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro," European Biophysics Journal, vol. 29, pp. 499-506, 2000.
[22]M.-T. Tsai, et al., "Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineering," Bioelectromagnetics, vol. 28, pp. 519-28, Oct 2007.
[23]K. Chang and W. H. S. Chang, "Pulsed electromagnetic fields prevent osteoporosis in an ovariectomized female rat model: a prostaglandin E2-associated process," Bioelectromagnetics, vol. 24, pp. 189-198, 2003.
[24]D. Jing, et al., "Circadian rhythm affects the preventive role of pulsed electromagnetic fields on ovariectomy-induced osteoporosis in rats," Bone, 2009.
[25]J. Manjhi, et al., "Effect of low level capacitive-coupled pulsed electric field stimulation on mineral profile of weight-bearing bones in ovariectomized rats," Journal of biomedical materials research. Part B, Applied biomaterials, 2009.
[26]I. Boutron, et al., "A checklist to evaluate a report of a nonpharmacological trial (CLEAR NPT) was developed using consensus," Journal of Clinical Epidemiology, vol. 58, pp. 1233-40, Dec 2005.
[27]J. D. Heckman and J. Sarasohn-Kahn, "The economics of treating tibia fractures. The cost of delayed unions," Bulletin of the Hospital for Joint Diseases, vol. 56, pp. 63-72, 1997.
[28]M. Hadjiargyrou, et al., "Enhancement of fracture healing by low intensity ultrasound," Clinical Orthopaedics & Related Research, pp. S216-29, Oct 1998.
[29]G. E. Friedlaender, et al., "Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions.[see comment]," Journal of Bone & Joint Surgery - American Volume, vol. 83-A Suppl 1, pp. S151-8, 2001.
[30]F. Lecanda, et al., "Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2," Journal of Cellular Biochemistry, vol. 67, pp. 386-96, Dec 1 1997.
[31]J. D. Rompe, et al., "High-energy extracorporeal shock wave treatment of nonunions," Clinical Orthopaedics & Related Research, pp. 102-11, Jun 2001.
[32]C.-J. Wang, et al., "Shock wave treatment shows dose-dependent enhancement of bone mass and bone strength after fracture of the femur," Bone, vol. 34, pp. 225-30, Jan 2004.
[33]M. W. Otter, et al., "Effects of electromagnetic fields in experimental fracture repair," Clinical orthopaedics and related research, vol. 355, p. S90, 1998.
[34]K.-S. Leung, et al., "Complex tibial fracture outcomes following treatment with low-intensity pulsed ultrasound," Ultrasound in Medicine & Biology, vol. 30, pp. 389-95, Mar 2004.
[35]E. Mayr, et al., "Ultrasound--an alternative healing method for nonunions?," Archives of Orthopaedic & Trauma Surgery, vol. 120, pp. 1-8, 2000.
[36]A. Emami, et al., "No effect of low-intensity ultrasound on healing time of intramedullary fixed tibial fractures," Journal of Orthopaedic Trauma, vol. 13, pp. 252-7, May 1999.
[37]L. Handolin, et al., "No long-term effects of ultrasound therapy on bioabsorbable screw-fixed lateral malleolar fracture," Scandinavian Journal of Surgery: SJS, vol. 94, pp. 239-42, 2005.
[38]A. A. Pilla, et al., "Non-invasive low-intensity pulsed ultrasound accelerates bone healing in the rabbit," Journal of Orthopaedic Trauma, vol. 4, pp. 246-53, 1990.
[39]S. J. Wang, et al., "Low intensity ultrasound treatment increases strength in a rat femoral fracture model," Journal of Orthopaedic Research, vol. 12, pp. 40-7, Jan 1994.
[40]K. H. Yang, et al., "The relationship between loading conditions and fracture patterns of the proximal femur," Journal of Biomechanical Engineering, vol. 118, pp. 575-8, Nov 1996.
[41]M. C. Fyfe and L. A. Chahl, "Mast cell degranulation and increased vascular permeability induced by 'therapeutic' ultrasound in the rat ankle joint," British Journal of Experimental Pathology, vol. 65, pp. 671-6, Dec 1984.
[42]D. K. Martin, et al., "Human macrophages contain a stretch-sensitive potassium channel that is activated by adherence and cytokines," Journal of Membrane Biology, vol. 147, pp. 305-15, Oct 1995.
[43]S. R. Young and M. Dyson, "Macrophage responsiveness to therapeutic ultrasound," Ultrasound in Medicine & Biology, vol. 16, pp. 809-16, 1990.
[44]F.-S. Wang, et al., "Nitric oxide mediates ultrasound-induced hypoxia-inducible factor-1alpha activation and vascular endothelial growth factor-A expression in human osteoblasts," Bone, vol. 35, pp. 114-23, Jul 2004.
[45]K. Ebisawa, et al., "Ultrasound enhances transforming growth factor beta-mediated chondrocyte differentiation of human mesenchymal stem cells," Tissue Engineering, vol. 10, pp. 921-9, May-Jun 2004.
[46]E. F. Sant'Anna, et al., "Effect of low intensity pulsed ultrasound and BMP-2 on rat bone marrow stromal cell gene expression," Journal of Orthopaedic Research, vol. 23, pp. 646-52, May 2005.
[47]C. T. Brighton, et al., "Fracture healing in the rabbit fibula when subjected to various capacitively coupled electrical fields," Journal of Orthopaedic Research, vol. 3, pp. 331-340, 1985.
[48]C. T. Brighton and S. R. Pollack, "Treatment of recalcitrant non-union with a capacitively coupled electrical field. A preliminary report," The Journal of Bone and Joint Surgery, vol. 67, p. 577, 1985.
[49]G. Scott and J. B. King, "A prospective, double-blind trial of electrical capacitive coupling in the treatment of non-union of long bones," The Journal of Bone and Joint Surgery, vol. 76, p. 820, 1994.
[50]R. J. Fitzsimmons, et al., "Embryonic bone matrix formation is increased after exposure to a low-amplitude capacitively coupled electric field, in vitro," Biochimica et Biophysica Acta, vol. 882, pp. 51-6, Jun 3 1986.
[51]H. Zhuang, et al., "Electrical stimulation induces the level of TGF-beta1 mRNA in osteoblastic cells by a mechanism involving calcium/calmodulin pathway," Biochemical & Biophysical Research Communications, vol. 237, pp. 225-9, Aug 18 1997.
[52]C. A. Bassett, et al., "Pulsing electromagnetic field treatment in ununited fractures and failed arthrodeses," JAMA, vol. 247, pp. 623-8, Feb 5 1982.
[53]R. J. Midura, et al., "Pulsed electromagnetic field treatments enhance the healing of fibular osteotomies," Journal of Orthopaedic Research, vol. 23, pp. 1035-1046, 2005.
[54]H. H. Guerkov, et al., "Pulsed electromagnetic fields increase growth factor release by nonunion cells," Clinical Orthopaedics & Related Research, pp. 265-79, Mar 2001.
[55]C. H. Lohmann, et al., "Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production," Journal of Orthopaedic Research, vol. 18, pp. 637-646, 2000.
[56]M.-T. Tsai, et al., "Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation," Journal of Orthopaedic Research, vol. 27, pp. 1169-74, Sep 2009.
[57]J. A. Buckwalter and H. J. Mankin, "Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation," Instructional Course Lectures, vol. 47, pp. 487-504, 1998.
[58]M. Brittberg, et al., "Carbon fiber scaffolds in the treatment of early knee osteoarthritis. A prospective 4-year followup of 37 patients," Clinical Orthopaedics & Related Research, pp. 155-64, Oct 1994.
[59]V. E. Hoikka, et al., "Reconstruction of the patellar articulation with periosteal grafts. 4-year follow-up of 13 cases," Acta Orthopaedica Scandinavica, vol. 61, pp. 36-9, Feb 1990.
[60]G. N. Homminga, et al., "Perichondral grafting for cartilage lesions of the knee," Journal of Bone & Joint Surgery - British Volume, vol. 72, pp. 1003-7, Nov 1990.
[61]M. Brittberg, et al., "Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.[see comment]," New England Journal of Medicine, vol. 331, pp. 889-95, Oct 6 1994.
[62]L. Peterson, "Articular cartilage injuries treated with autologous chondrocyte transplantation in the human knee," Acta Orthopaedica Belgica, vol. 62 Suppl 1, pp. 196-200, 1996.
[63]P. S. J. M. Bouwmeester, et al., "A retrospective analysis of two independent prospective cartilage repair studies: autogenous perichondrial grafting versus subchondral drilling 10 years post-surgery," Journal of Orthopaedic Research, vol. 20, pp. 267-73, Mar 2002.
[64]E. Kon, et al., "Second generation issues in cartilage repair," Sports Medicine & Arthroscopy Review, vol. 16, pp. 221-9, Dec 2008.
[65]T. Minas and S. Nehrer, "Current concepts in the treatment of articular cartilage defects," Orthopedics, vol. 20, pp. 525-38, Jun 1997.
[66]S. P. Bruder, et al., "Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells," Journal of Orthopaedic Research, vol. 16, pp. 155-62, Mar 1998.
[67]J. Goshima, et al., "The origin of bone formed in composite grafts of porous calcium phosphate ceramic loaded with marrow cells," Clinical Orthopaedics & Related Research, pp. 274-83, Aug 1991.
[68]C. Maniatopoulos, et al., "Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats," Cell & Tissue Research, vol. 254, pp. 317-30, Nov 1988.
[69]H. Nakahara, et al., "Bone and cartilage formation in diffusion chambers by subcultured cells derived from the periosteum," Bone, vol. 11, pp. 181-8, 1990.
[70]H. S. Cheung, et al., "In vitro synthesis of tissue-specific type II collagen by healing cartilage. I. Short-term repair of cartilage by mature rabbits," Arthritis & Rheumatism, vol. 23, pp. 211-9, Feb 1980.
[71]T. Furukawa, et al., "Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee," Journal of Bone & Joint Surgery - American Volume, vol. 62, pp. 79-89, Jan 1980.
[72]N. Selvamurugan, et al., "Effects of BMP-2 and pulsed electromagnetic field (PEMF) on rat primary osteoblastic cell proliferation and gene expression," Journal of Orthopaedic Research, vol. 25, pp. 1213-20, Sep 2007.
[73]C. W. Chen, et al., "Type I and II collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells," Journal of Orthopaedic Research, vol. 23, pp. 446-53, Mar 2005.
[74]B. Johnstone, et al., "In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells," Experimental Cell Research, vol. 238, pp. 265-72, Jan 10 1998.
[75]T. A. Linkhart, et al., "Growth factors for bone growth and repair: IGF, TGF beta and BMP," Bone, vol. 19, pp. 1S-12S, Jul 1996.
[76]S. J. Warden, et al., "Low-intensity pulsed ultrasound stimulates a bone-forming response in UMR-106 cells," Biochemical & Biophysical Research Communications, vol. 286, pp. 443-50, Aug 24 2001.
[77]T. Takayama, et al., "Low-intensity pulsed ultrasound stimulates osteogenic differentiation in ROS 17/2.8 cells," Life Sciences, vol. 80, pp. 965-71, Feb 13 2007.
[78]B. H. Choi, et al., "Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture," Journal of Biomedical Materials Research, vol. Part A. 79, pp. 858-64, Dec 15 2006.
[79]S. D. Cook, et al., "Improved cartilage repair after treatment with low-intensity pulsed ultrasound," Clinical Orthopaedics & Related Research, pp. S231-43, Oct 2001.
[80]J. Parvizi, et al., "Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression," Journal of Orthopaedic Research, vol. 17, pp. 488-94, Jul 1999.
[81]Z.-J. Zhang, et al., "The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production," Ultrasound in Medicine & Biology, vol. 29, pp. 1645-51, Nov 2003.
[82]J. D. Heckman, et al., "Acceleration of tibial fracture-healing by non-invasive, low-intensity pulsed ultrasound," Journal of Bone & Joint Surgery - American Volume, vol. 76, pp. 26-34, Jan 1994.
[83]P. H. W. Lubbert, et al., "Low-intensity pulsed ultrasound (LIPUS) in fresh clavicle fractures: a multi-centre double blind randomised controlled trial," Injury, vol. 39, pp. 1444-52, Dec 2008.
[84]S. Rutten, et al., "Use of low-intensity pulsed ultrasound for posttraumatic nonunions of the tibia: a review of patients treated in the Netherlands," Journal of Trauma-Injury Infection & Critical Care, vol. 62, pp. 902-8, Apr 2007.
[85]X.-l. Jia, et al., "Effects of low-intensity pulsed ultrasound in repairing injured articular cartilage," Chinese Journal of Traumatology, vol. 8, pp. 175-8, Jun 2005.
[86]L. K. Papatheodorou, et al., "Effect of transosseous application of low-intensity ultrasound at the tendon graft-bone interface healing: gene expression and histological analysis in rabbits," Ultrasound in Medicine & Biology, vol. 35, pp. 576-84, Apr 2009.
[87]H. J. Lee, et al., "Low-intensity ultrasound inhibits apoptosis and enhances viability of human mesenchymal stem cells in three-dimensional alginate culture during chondrogenic differentiation," Tissue Engineering, vol. 13, pp. 1049-57, May 2007.
[88]H. J. Lee, et al., "Low-intensity ultrasound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells," Artificial Organs, vol. 30, pp. 707-15, Sep 2006.
[89]D. Schumann, et al., "Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound enhances the chondrogenic phenotype in vitro," Biorheology, vol. 43, pp. 431-43, 2006.
[90]P. M. van der Kraan and W. B. van den Berg, "Osteophytes: relevance and biology," Osteoarthritis & Cartilage, vol. 15, pp. 237-44, Mar 2007.
[91]C.-W. D. Chen, et al., "sAPPalpha enhances the transdifferentiation of adult bone marrow progenitor cells to neuronal phenotypes," Current Alzheimer Research, vol. 3, pp. 63-70, Feb 2006.
[92]Y. Jiang, et al., "Pluripotency of mesenchymal stem cells derived from adult marrow.[see comment][erratum appears in Nature. 2007 Jun 14;447(7146):879-80]," Nature, vol. 418, pp. 41-9, Jul 4 2002.
[93]A. Gleizal, et al., "Transcriptional expression of calvarial bone after treatment with low-intensity ultrasound: an in vitro study," Ultrasound in Medicine & Biology, vol. 32, pp. 1569-74, Oct 2006.
[94]J. G.-R. Li, et al., "Optimum intensities of ultrasound for PGE(2) secretion and growth of osteoblasts," Ultrasound in Medicine & Biology, vol. 28, pp. 683-90, May 2002.
[95]J. K. Li, et al., "Cytokine release from osteoblasts in response to ultrasound stimulation," Biomaterials, vol. 24, pp. 2379-85, Jun 2003.
[96]C. B. Goodwin, et al., "A double-blind study of capacitively coupled electrical stimulation as an adjunct to lumbar spinal fusions," Spine, vol. 24, p. 1349, 1999.
[97]J. H. Cui, et al., "Effects of low-intensity ultrasound on chondrogenic differentiation of mesenchymal stem cells embedded in polyglycolic acid: an in vivo study," Tissue Engineering, vol. 12, pp. 75-82, Jan 2006.
[98]J. H. Cui, et al., "Preconditioning of mesenchymal stem cells with low-intensity ultrasound for cartilage formation in vivo," Tissue Engineering, vol. 13, pp. 351-60, Feb 2007.
[99]H. H. Cho, et al., "Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors," Journal of Cellular Biochemistry, vol. 96, pp. 533-42, Oct 15 2005.
[100]R. Yagi, et al., "Intrajoint comparisons of gene expression patterns in human osteoarthritis suggest a change in chondrocyte phenotype," Journal of Orthopaedic Research, vol. 23, pp. 1128-38, Sep 2005.
[101]N. Miosge, et al., "Expression of collagen type I and type II in consecutive stages of human osteoarthritis," Histochemistry & Cell Biology, vol. 122, pp. 229-36, Sep 2004.
[102]Z. Chen, et al., "Gap junction protein connexin 43 serves as a negative marker for a stem cell-containing population of human limbal epithelial cells," Stem Cells, vol. 24, pp. 1265-73, May 2006.
[103]D. L. Diefenderfer, et al., "Regulation of BMP-induced transcription in cultured human bone marrow stromal cells," Journal of Bone & Joint Surgery - American Volume, vol. 85-A Suppl 3, pp. 19-28, 2003.
[104]K. L. Ou, et al., "Effects of the nanostructure and nanoporosity on bioactive nanohydroxyapatite/reconstituted collagen by electrodeposition," J Biomed Mater Res A, Mar 12 2009.
[105]G. Friedl, et al., "Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro," Osteoarthritis & Cartilage, vol. 15, pp. 1293-300, Nov 2007.
[106]S. Rutten, et al., "Low-intensity pulsed ultrasound increases bone volume, osteoid thickness and mineral apposition rate in the area of fracture healing in patients with a delayed union of the osteotomized fibula," Bone, vol. 43, pp. 348-54, Aug 2008.
[107]S. W. O'Driscoll, et al., "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," Journal of Bone & Joint Surgery - American Volume, vol. 70, pp. 595-606, Apr 1988.
[108]S. C. Chen, et al., "Increases in bone mineral density after functional electrical stimulation cycling exercises in spinal cord injured patients," Disability & Rehabilitation.27(22):1337-41, 2005.
[109]A. E. Grigoriadis, et al., "Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of clonal chondrogenic rat cell lines," Differentiation, vol. 60, pp. 299-307, Sep 1996.
[110]P. M. van der Kraan, et al., "TGF-beta signaling in chondrocyte terminal differentiation and osteoarthritis: modulation and integration of signaling pathways through receptor-Smads," Osteoarthritis & Cartilage, vol. 17, pp. 1539-45, Dec 2009.
[111]R. F. Loeser, et al., "Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen," Osteoarthritis & Cartilage, vol. 8, pp. 96-105, Mar 2000.
[112]M. J. Humphries, "The molecular basis and specificity of integrin-ligand interactions," Journal of Cell Science, vol. 97, pp. 585-92, Dec 1990.
[113]R. O. Hynes, "Integrins: versatility, modulation, and signaling in cell adhesion," Cell, vol. 69, pp. 11-25, Apr 3 1992.
[114]R. O. Hynes, "Integrins: bidirectional, allosteric signaling machines," Cell, vol. 110, pp. 673-87, Sep 20 2002.
[115]J. Hanai, et al., "Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter," Journal of Biological Chemistry, vol. 274, pp. 31577-82, Oct 29 1999.
[116]A. Zwijsen, et al., "New intracellular components of bone morphogenetic protein/Smad signaling cascades," FEBS Letters, vol. 546, pp. 133-9, Jul 3 2003.
[117]A. Chantraine, et al., "Bone remodeling during the development of osteoporosis in paraplegia," Calcified Tissue International, vol. 38, pp. 323-7, Jun 1986.
[118]J. F. Keating, et al., "Minimal trauma causing fractures in patients with spinal cord injury," Disability & Rehabilitation.14(2):108-9, p. Jun, 1992.
[119]S.-D. Jiang, et al., "Osteoporosis after spinal cord injury.[erratum appears in Osteoporos Int. 2006;17(8):1278-81]," Osteoporosis International, vol. 17, pp. 180-92, Feb 2006.
[120]W. M. Nottage, "A review of long-bone fractures in patients with spinal cord injuries," Clinical Orthopaedics & Related Research, pp. 65-70, Mar-Apr 1981.
[121]K. K. BeDell, et al., "Effects of functional electrical stimulation-induced lower extremity cycling on bone density of spinal cord-injured patients," American Journal of Physical Medicine & Rehabilitation.75(1):29-34, p. Feb, 1996.
[122]E. M. Leeds, et al., "Bone mineral density after bicycle ergometry training," Archives of Physical Medicine & Rehabilitation.71(3):207-9, 1990.
[123]T. Mohr, et al., "Increased bone mineral density after prolonged electrically induced cycle training of paralyzed limbs in spinal cord injured man," Calcified Tissue International.61(1):22-5, 1997.
[124]N. E. Naftchi, et al., "Mineral metabolism in spinal cord injury," Archives of Physical Medicine & Rehabilitation, vol. 61, pp. 139-42, Mar 1980.
[125]D. Roberts, et al., "Longitudinal study of bone turnover after acute spinal cord injury," Journal of Clinical Endocrinology & Metabolism, vol. 83, pp. 415-22, Feb 1998.
[126]B. J. Kiratli, et al., "Bone mineral and geometric changes through the femur with immobilization due to spinal cord injury," Journal of Rehabilitation Research & Development, vol. 37, pp. 225-33, Mar-Apr 2000.
[127]E. Wilmet, et al., "Longitudinal study of the bone mineral content and of soft tissue composition after spinal cord section," Paraplegia, vol. 33, pp. 674-7, Nov 1995.
[128]P. Vestergaard, et al., "Fracture rates and risk factors for fractures in patients with spinal cord injury," Spinal Cord, vol. 36, pp. 790-6, Nov 1998.
[129]F. Biering-Sorensen, et al., "Non-pharmacological treatment and prevention of bone loss after spinal cord injury: a systematic review," Spinal Cord, Jan 27 2009.
[130]P. Eser, et al., "Effect of electrical stimulation-induced cycling on bone mineral density in spinal cord-injured patients," European Journal of Clinical Investigation, vol. 33, pp. 412-9, May 2003.
[131]F. M. Maynard, Jr., et al., "International Standards for Neurological and Functional Classification of Spinal Cord Injury. American Spinal Injury Association," Spinal Cord, vol. 35, pp. 266-74, May 1997.
[132]Y. N. Chen HY, Chen KS, Tsai KH, Fu LL, Chen SC, H MH, Chen JJJ, "Development of FES-cycling System with Network Capability for Multi-Center Clinical Studies," J. Med. Biol. Eng., vol. 21, pp. 85-92, 2001.
[133]J. J. Chen, et al., "Applying fuzzy logic to control cycling movement induced by functional electrical stimulation," IEEE Transactions on Rehabilitation Engineering, vol. 5, pp. 158-69, Jun 1997.
[134]R. B. Mazess and H. S. Barden, "Measurement of bone by dual-photon absorptiometry (DPA) and dual-energy X-ray absorptiometry (DEXA). [Review] [50 refs]," Annales Chirurgiae et Gynaecologiae.77(5-6):197-203, 1988.
[135]A. Goulding, et al., "Changing femoral geometry in growing girls: a cross-sectional DEXA study," Bone, vol. 19, pp. 645-9, Dec 1996.
[136]A. Frotzler, et al., "Effect of detraining on bone and muscle tissue in subjects with chronic spinal cord injury after a period of electrically-stimulated cycling: a small cohort study," Journal of Rehabilitation Medicine, vol. 41, pp. 282-5, Mar 2009.
[137]A. F. Stewart, et al., "Calcium homeostasis in immobilization: an example of resorptive hypercalciuria," New England Journal of Medicine.306(19):1136-40, 1982.
[138]L. Maimoun, et al., "Changes in osteoprotegerin/RANKL system, bone mineral density, and bone biochemicals markers in patients with recent spinal cord injury," Calcified Tissue International, vol. 76, pp. 404-11, Jun 2005.
[139]B. J. Kiratli, et al., "Bone mineral and geometric changes through the femur with immobilization due to spinal cord injury," Journal of Rehabilitation Research & Development.37(2):225-33, p. Apr, 2000.
[140]L. Schultheis, "The mechanical control system of bone in weightless spaceflight and in aging," Experimental Gerontology, vol. 26, pp. 203-14, 1991.
[141]M. Belanger, et al., "Electrical stimulation: can it increase muscle strength and reverse osteopenia in spinal cord injured individuals?," Archives of Physical Medicine & Rehabilitation, vol. 81, pp. 1090-8, Aug 2000.
[142]W. Phillips, et al., "Relative changes in blood flow with functional electrical stimulation during exercise of the paralyzed lower limbs," Paraplegia, vol. 33, pp. 90-3, Feb 1995.
[143]T. Hagiwara and W. H. Bell, "Effect of electrical stimulation on mandibular distraction osteogenesis," Journal of Cranio-Maxillo-Facial Surgery, vol. 28, pp. 12-9, Feb 2000.
[144]A. Hartkopp, et al., "Bone fracture during electrical stimulation of the quadriceps in a spinal cord injured subject," Archives of Physical Medicine & Rehabilitation, vol. 79, pp. 1133-6, Sep 1998.
[145]M. Dauty, et al., "Supralesional and sublesional bone mineral density in spinal cord-injured patients," Bone, vol. 27, pp. 305-9, Aug 2000.
[146]P. Eser, et al., "Fracture threshold in the femur and tibia of people with spinal cord injury as determined by peripheral quantitative computed tomography," Archives of Physical Medicine & Rehabilitation, vol. 86, pp. 498-504, Mar 2005.
[147]D. E. Garland, et al., "Risk factors for osteoporosis at the knee in the spinal cord injury population.[see comment]," Journal of Spinal Cord Medicine, vol. 27, pp. 202-6, 2004.