富含血小板纖維蛋白對肌肉、骨骼及椎間盤再生之作用

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Title:	富含血小板纖維蛋白對肌肉、骨骼及椎間盤再生之作用
Other Titles:	Effects of Platelet-Rich Fibrin (PRF) on Regeneration of Muscle, Bone, and Intervertebral Disc
Authors:	劉耿彰 LIU, KENG-CHANG
Contributors:	楊錫坤、歐柏榮 YANG, SHYI-KUEN;OU, BOR-RUNG 畜產與生物科技學系
Keywords:	富含血小板纖維蛋白;肌母細胞;纖維母細胞;噬骨細胞;內皮細胞;脊椎融合;椎間盤;盤源性背痛;脊椎內視鏡 Platelet-rich fibrin (PRF);Myoblast;Fibroblast;Osteoclast;Endothelial cells;Spinal fusion surgery;Disc;Endoscopic spine surgery;Discogenic back pain
Date:	2019
Issue Date:	2019-12-16T04:03:33Z (UTC)
Abstract:	血小板內含多種活性生長因子，釋放後可以促進血管增生及組織的再生與修復。一般組織中，血小板的濃度不夠高，而且混雜其間的紅血球及白血球可能對組織的修復產生負面的影響。血小板濃縮物，包括富含血小板血漿（platelet-rich plasma; PRP）和富含血小板纖維蛋白（platelet-rich fibrin; PRF），是藉由特定的處理程序取到純化且高濃度的血小板製品，內含更豐富的生長因子，因此理論上更能夠促進組織修復及生長。PRP 是抽取自體的血液，經儀器離心處理後，取中間層富含血小板的血漿，再重新注射回體內。PRP在臨床的使用範圍相當廣泛，在骨科的領域裡最常用於局部肌腱、韌帶或關節的治療。PRF是第二代的血小板濃縮技術，與PRP比較，PRF製備時無需添加任何生物製劑即可獲得富含血小板及生長因子的纖維蛋白，臨床上可當再生膜使用，而有助於組織缺損的修復。然而，有關PRF對於肌肉、骨骼及椎間盤生物性作用的資料有限。本研究包含三大主題，第一主題是探討PRF對於四種不同細胞之增殖與分化的作用，包括大鼠肌母細胞 (L8)、噬骨細胞的前驅RAW 264.7單核細胞、L929纖維母細胞、和PEC內皮細胞。我們分別將這四種細胞加入PRF共同培養，來模擬組織中的細胞生長實驗，並以馬的血清當對照組。結果顯示PRF對L8肌母細胞和L929纖維母細胞有負面的調節作用，而且細胞週期的調控應在第零間期 (G0 phase)。相反的，PRF 對RAW 264.7單核細胞和PEC內皮細胞卻有正面的調節作用。我們再探討不同濃度釋放液對L8細胞增殖的影響，結果顯示含有高濃度釋放液組別的肌母細胞數目反而較低濃度釋放液的組別為少。我們推測 PRF的作用可能有細胞種類和濃度的差別。第二主題是探討PRF在臨床脊椎融合手術中，對脊椎融合的效益。脊椎後側椎間融合術常被運用在治療脊椎部分的疾病，例如滑脫、畸形、腫瘤等，而骨融合率決定了手術的成功率。目前移植骨骼的金標準是自體骨骼，因為有最好的骨融合率。我們將取自骨銀行的異體骨混合自體PRF和自體骨髓置入準備好的椎間盤間隙中，一年的骨融合率大於 90%，比使用異體骨骼為好，與完全使用自體骨骼相比，融合率則無顯著差異。這顯示PRF可促進脊椎融合手術中使用異體骨的融合率，但手術時間縮短，出血量較少，滿意度較高，因為節省了採自體骨的時間和避免取骨所衍生的後遺症。第三主題是探討PRF對椎間盤修復的效果。在脊椎的病變中，沒有神經症狀的嚴重背痛一直是很棘手的議題，不僅痛源難以診斷，治療的方式也頗有爭議。盤源性背痛，是指因椎間盤退化或發炎所導致的背痛，佔了其中六到八成，我們首先利用核磁共振和激發式椎間盤照影確定盤源性背痛的診斷，並選擇適當的病人，然後採用微創經皮脊椎內視鏡手術處理這些患者椎間盤的病變處。結果證實有很高的成功率和滿意度，其中成功的關鍵在於徹底移除椎間盤中發炎的纖維環和退化的髓核。在之後更進一步的臨床試驗中，我們再將自體PRF置入六位患者因盤源性背痛接受經皮脊椎內視鏡減壓和清創手術的椎間盤內，觀察臨床結果，並利用影像學觀察椎間盤一年的變化。結果發現六位中的五位有最高的滿意度，並且置入PRF的椎間盤，核磁共振影像訊號未再顯示更進一步的退化，由此推斷PRF有修復椎間盤的效果。 Platelet concentrates including platelet-rich plasma (PRP) andplatelet-rich fibrin (PRF) are becoming innovative tools of regenerativemedicine. Autologous PRP is derived from an individual's whole bloodthen centrifuged to remove red blood cells and leucocytes. Theremaining plasma has a 5- to 10-fold higher concentration of growthfactors than whole blood. These growth factors have been found topromote natural healing responses, and hence PRP is widely used inorthopedic and sports medicine to relieve pain in musculoskeletalinjuries. Platelet-rich fibrin (PRF) is a newer generation of plateletconcentrates and has being used as an adjunctive autologousbiomaterial to promote bone and soft tissue healing and regeneration.This thesis included an in vitro study and 3 clinical trials, and theobjectives were to investigate the roles of PRF on regeneration of muscle,tendon, bone, and intervertebral disc (IVD). In vitro study, weinvestigated the biologic effects of pig PRF on the proliferation ofdifferent cells, including rat L8 myoblasts, mouse RAW 264.7 monocyticcells which are precursors of osteoclasts, mouse L929 fibroblasts, andporcine endothelial cells (PEC). L8 cells and L929 cells cultured with PRFshowed a decrease in cell number when compared to the control groupin which horse serum was used as a control. RAW 264.7 cells and PECcultured with PRF, on the other hand, increased cell number. Theseresults suggested that PRF had negative effect on the proliferation of L8and L929 cells, however, expressed positive regulation on RAW 264.7cells and PEC. In clinical trials, we investigated the clinical effects of PRFon bone fusion in spinal fusion surgery and IVD regeneration afterendoscopic discectomy. Posterior lumbar decompression and 2transforaminal interbody fusion (TLIF) is a safe and effective surgery fordegenerative spinal diseases. Several options of bone graft are availablebut autologous iliac crest bone grafting (ICBG) is still the gold standard.However, some morbidity is associated with ICBG harvesting such asinfection, fracture, or donor site pain. To avoid donor site morbidity,various allograft materials have been used as a substitute but are stillquestionable as to the effectiveness and safety. In our study, allograftcombined with autogenous bone marrow and with/without PRF wasused to compare with autologous ICBG in TLIF surgery. After at least 12months follow-up, the fusion rate in single level TLIF was 100% in bothautograft group and allograft with PRF group, while the fusion rate ofallograft without PRF was 77.8%. The results demonstrated positiveeffect of PRF on bone fusion in TLIF. Discogenic low back pain (DLBP) isassociated with degeneration of IVD and usually is a complex problemregrading to diagnosis and treatment. It is suspected that the pain sourceof DLBP comes from the torn lesion of annulus fibrosus (AF) which islocated on the outer surface of IVD. We applied percutaneousendoscope to treat torn AF in patients with DLBP and the satisfactoryoutcomes were more than 85% in selected patients at two yearsfollow-up. The procedure is effective and minimally invasive becauseonly inflammatory tissues on AF and loose nucleus debris are removedunder clear endoscopic vision. To investigate the effect of PRF onregeneration of IVD, we inserted PRF into IVD in 6 patients who werediagnosed with DLBP and had undergone endoscopic treatment.Autogenous PRF was inserted into nucleus at the end of endoscopicprocedure. At 12 months follow-up, 5 of 6 patients showed excellentresults and MRI T2-weighted signal of involved IVD did not show furtherdegenerative change. This adjunctive biomaterial expressed beneficialeffects on clinical outcomes and radiologic results.
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