血小板富含血漿合併膠原蛋白基質於關節炎軟骨細胞再生之研究

The regeneration potential of the combination of platelet-rich plasma and collagen matrix in an arthritic chondrocyte cell model

吳佳哲(Chia-Che Wu)

指導教授 ： 吳志雄

共同指導教授 ： 鄧文炳(Win-Ping Deng)

臺北醫學大學/醫學院/臨床醫學研究所/博士(2011年)

https://doi.org/10.6831/TMU.2011.00164

摘要

本研究是評估膠原基質加強富含血小板血漿（PRP）影響促炎性細胞激素誘導關節發炎模型的角色。我們之前已經證實富含血小板血漿於恢復椎間盤退化和骨質疏鬆的高度再生作用。在這項研究中，膠原基質調控的富含血小板血漿作為再生和反炎症介質來挽救促炎症介白質IL- 1β（10ng/ml）及腫瘤壞死因子-α（20ng/ml）引起的軟骨細胞退化。首先，細胞活性測試法結果顯示，含1 ng/ml轉化生長因子-β1之富含血小板血漿為最佳軟骨細胞增殖劑量。富含血小板血漿可挽救介白質IL- 1β加上腫瘤壞死因子-α抑制下的軟骨細胞特異性基因表現，尤其是在膠原基質的調控下。被介白質IL - 1β加上腫瘤壞死因子-α激活的發炎因子也因富含血小板血漿含膠原基質的處理而顯著減少。由介白質IL-1β加上腫瘤壞死因子-α造成的強烈抑制細胞膜受體integrin α1β1和CD44的作用，也因富含血小板血漿濃厚液加膠原塗層而恢復。在一個封裝膠原蛋白的三維模型裡，富含血小板血漿高度增強誘導軟骨新生，如加強恢復介白質IL- 1β加上腫瘤壞死因子-α抑制的第二型膠原蛋白和聚醣。此結果表示，膠原基質可增強富含血小板血漿恢復促發炎反應細胞激素抑制軟骨新生的效應。這種結合富含血小板血漿和膠原基質的生理微環境可能有利於促進維持軟骨細胞的生理動態平衡，並可發展先進的骨關節炎療法且應用於臨床。
另外，小耳症是因不完整發展的外耳而形成畸形小耳，治療上的重建手術仍然有限且需要再開發。基於上述研究，延伸運用富含血小板血漿濃厚液培養小耳軟骨細胞形成新的耳廓軟骨以供將來的耳廓重建使用。
因此，富含血小板血漿被當做生長因子以促進體外小耳軟骨再生。小耳軟骨細胞在含膠原蛋白及富含血小板血漿的懸浮液內三維培養 4週。然後，從小耳症患者取得小耳軟骨細胞。同時，富含血小板血漿以轉化生長因子-β1的濃度來量化。小耳軟骨細胞於富含血小板血漿的單層和三維培養基中培養。 4週後，將人類組織工程耳廓軟骨利用蘇木紫-伊紅染色法(H＆E染色)及免疫組織化學法染第二型膠原蛋白以組織學分析。研究顯示，小耳軟骨細胞在富含血小板血漿於轉化生長因子-β1 750 pg / ml和1ng/ml的濃度下體外培養 9天可增加細胞的活性(cell viability)。體外小耳軟骨細胞合併富含血小板血漿培養21天後，從逆轉錄聚合酶鏈反應（RT - PCR）的結果顯示，表示軟骨細胞特異性基因的聚醣和第二型膠原蛋白均顯著且持續表現。小耳軟骨培養於富含血小板血漿/膠原支架可形成新的組織工程耳軟骨。於4週的培養後，以免疫組織化學法染第二型膠原蛋白，顯示信號密集於細胞和基質之間。
我們的研究結果顯示，富含血小板血漿促進小耳軟骨細胞的增殖和分化以增加再生潛能，組織再生之新生軟骨將來可運用於耳廓軟骨重建。

關鍵字

骨關節炎 ； 富含血小板血漿 ； 膠原基質 ； 促炎性細胞激素 ； 生理微環境 ； 小耳症 ； 新生軟骨

並列摘要

This study was undertaken to evaluate the role of collagen matrix to enhance platelet-rich plasma (PRP) effects on pro-inflammatory cytokine-induced arthritic model. We have previously demonstrated the highly regenerative roles of PRP to restore disc degeneration and osteoporosis. In this study, PRP modulated by collagen matrix was used as a regenerative and anti-inflammatory mediator to rescue the chondrocyte degeneration induced by pro-inflammatory cytokines IL-1???n(10ng/ml)+TNF-???n(20ng/ml). First, the MTT result indicated that 1 ng/ml TGF-??1 in PRP showed an optimal dosage for chondrocytes proliferation. The chondrogenic-specific gene expressions were rescued by PRP from the inhibition of IL-1??+TNF-??, especially under the modulation of collagen matrix. The inflammatory molecules activated by IL-1??+TNF-?? were also significantly diminished by PRP with collagen matrix. The membrane receptors integrin ??1??1 and CD44 were strongly inhibited by IL-1??+TNF-??, while this inhibition was then recovered by PRP in collagen coating condition. In a 3D model encapsulated with collagen, PRP-induced chondrogenesis were highly enhanced, such as strong restoration of type II collagen and proteoglycan from the inhibition of IL-1??+TNF-??. The result indicated that collagen matrix enhances the effect of PRP on chondrogenesis in response to pro-inflammatory cytokines. The combination of PRP and collagen matrix might facilitate a physiological microenvironment beneficial for maintaining chondrocyte homeostasis and represents an advanced osteoarthritis therapy for clinical applications.
Besides, microtia is suggested as an incomplete auricular development and surgical reconstruction for microtia is still limited and emergent to be developed. Base on the previous study, further research was aimed to apply PRP for human auricular neo-cartilage reconstruction by using microtia chondrocytes.
So PRP was used as growth factor source to promote in vitro microtia regeneration. Microtia chondrocytes were then suspended in collagen for 3D culture for 4 weeks with PRP. Then chondrocytes were obtained from microtia patient. Meanwhile, PRP was extracted and quantified by transforming growth factor-???n1 (TGF-??1) concentration. Microtia chondrocytes were then cultured with PRP in monolayer and 3-dimentional (3D) cultures. After 4 weeks, human engineered auricular cartilage was then histologically analyzed by using H&E staining and immunohistochemistry (IHC) of type II collagen (Col II). It is demonstrated that the PRP (TGF-??1 750 pg/ml and 1ng/ml) increased cell viability of microtia chondrocyte during in vitro 9-day cultures. From the reverse-transcriptase polymerase chain reaction (RT-PCR) results, chondrogenic-specific mRNA of aggrecan and type II collagen (Col II) were significantly and continuously expressed by PRP during 21-day in vitro expansion. Tissue-engineered auricular neo-cartilage was performed by seeding microtia chondrocytes in PRP/ collagen scaffold. Immunohistochemistry (IHC) of Col II showed intensive signals between cells and matrix after 4-week cultures.
Our results demonstrated that PRP promotes proliferation and redifferentiation of microtia chondrocytes and provides regenerative potentials of auricular neo-cartilage reconstruction.

並列關鍵字

Osteoarthritis (OA) ； Platelet-rich plasma (PRP) ； Collagen matrix ； Physiological microenvironment ； Pro-inflammatory cytokines ； Microtia ； Neo-cartilage

參考文獻

1. Yang Y: Skeletal morphogenesis during embryonic development. Crit Rev Eukaryot Gene Expr 2009, 19(3):197-218.

2. Karsenty G, Wagner EF: Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002, 2(4):389-406.

3. Miura M, Miura Y, Sonoyama W, Yamaza T, Gronthos S, Shi S: Bone marrow-derived mesenchymal stem cells for regenerative medicine in craniofacial region. Oral Dis 2006, 12(6):514-522.

4. Bertrand J, Cromme C, Umlauf D, Frank S, Pap T: Molecular mechanisms of cartilage remodelling in osteoarthritis. Int J Biochem Cell Biol 2010, 42(10):1594-1601.

5. Kirkham SG, Samarasinghe RK: Review article: Glucosamine. J Orthop Surg (Hong Kong) 2009, 17(1):72-76.