以富含血小板纖維蛋白(PRF)釋放液伴隨骨髓幹細胞治療骨質疏鬆症之成效：小鼠模式

Abstract

骨質疏鬆症是一種漸進性疾病，其特徵是骨骼組織的質量和數量異常，進而導致骨骼強度受損和易增加骨折的風險。雖然詳細的病理機制還需要更進一步去研究探討，但大致上可將骨質疏鬆症歸咎於雌性激素分泌降低，使得原本成骨細胞和破骨細胞之間保持的平衡急劇失衡，造成了骨質疏鬆症的發生。 目前骨質疏鬆症的治療主要是著重在降低骨質耗損速率或是促進新骨質的生成，使用的藥物包括了雙磷酸鹽、降鈣素、雌激素和激素替代療法（HRT）…等。雖然這些藥物現在仍然在臨床上使用以預防或治療骨質疏鬆症，但往往有較大的副作用。例如HRT可以預防骨質疏鬆症，但這些藥物卻會刺激子宮內膜增生，增加罹患子宮內膜癌或乳腺癌的風險。因此目前迫切需要開發用於預防和治療骨質疏鬆的替代方法。 幹細胞是一種未分化的細胞，可以無限分裂成原始幹細胞系，也還有分化成其它類型細胞之潛能。我們收集了小鼠腿骨的骨髓幹細胞，經過流式細胞儀篩選後，於誘導分化為成骨細胞的環境中培養。 富血小板纖維蛋白（PRF）是一種天然生長因子的來源，裡面含有大量會影響骨質再生的生長因子。包含:TGFb-1(Transforming growth factor)、PDGF (platelet-derived growthfactor)、VEGF (vascular endothelial growth factor)及Matrix proteins (thrombospondin-1、fibronectin)。這些PRF所釋放的生長因子，會透過促進細胞增生、基質形成、骨質生成、膠原蛋白的合成等。最近研究顯示，富血小板纖維蛋白（PRF）會非常緩慢地釋放出生長因子至少持續7天甚至最長達28天，這意味著PRF可以在傷口癒合過程中對周圍環境造成長時間的刺激影響。 我們將富含血小板纖維蛋白釋放液（PRFr）添加在進行培養的小鼠骨髓幹細胞，以探討釋放液是否能提高幹細胞的增殖能力及促進細胞分化潛能。結果顯示加入釋放液組別的骨髓幹細胞增殖速率較快速，細胞存活率也高出許多。因此我們推斷富含血小板纖釋放液對骨髓幹細胞生長及分化有極大幫助。 在完成細胞實驗之後，我們接著利用動物實驗，去探討富含血小板纖釋放液對骨質疏鬆症之治療成效。骨質疏鬆症模型建立是利用卵巢切除（OVX）小鼠， 16週齡的 ICR小鼠隨機分配至不手術、卵巢切除或假手術三個類別，不手術組也就是做為Pos.Control的正常小鼠，假手術組是將皮膚劃開後再縫合回去，不摘除卵巢，卵巢摘除組則在卵巢摘除後，分別接受八種不同的治療方法:（1）對照組:不注射任何物質，作為Neg.Control;（2）注射ㄧ次BMSCs組 3x105 BMSCs/0.6ml PBS（3）注射ㄧ次PRFr組，0.6ml PRFr ;（4）注射ㄧ次BMSCs+ PRFr組，3x105 BMSCs/0.6ml PRFr;（5）注射四次BMSCs組 1.2x106 BMSCs/0.6ml PBS ;（6）注射四次PRFr組，2.4ml PRFr;（7）注射四次BMSCs+ PRFr組，1.2x106 BMSCs/2.4ml PRFr ;（8）骨內注射四次BMSCs+ PRFr組，1.2x106 BMSCs/2.4ml PRFr。所使用的骨髓間葉幹細胞是從健康同種異體小鼠取得，而富含血小板纖維蛋白釋放液PRFr則是從健康兔子抽取血液製備而成。治療時將幹細胞或富含血小板纖維蛋白釋放液注射至小鼠的薦尾靜脈中，以調查其用於骨量恢復與骨質再生的治療潛力。 完成注射後之小鼠會再飼養八週，使其骨骼完整生長，最後將其犧牲並利用Micro CT 血清檢測 以及組織切片染色去評估治療成效。Micro CT掃描部位為左腿脛骨近端生長板下0.5~1.5mm處，分析項目包含骨礦物質密度、骨組織百分比、骨小樑數目、骨小樑厚度及骨小樑間距。分析後將腿骨先利用中性脫鈣液脫鈣，在以石蠟包埋切片，並染H&E染劑以放置於顯微鏡下觀察。 由實驗結果得之，僅僅注射一次BMSC或是PRF的治療小鼠，皆無明顯治療效果，骨質疏鬆症現象和不治療組小鼠相似。而注射四次BMSC或是PRF的治療小鼠，骨礦物質密度、骨組織百分比、骨小樑數目、骨小樑厚度及骨小樑間距等數據都明顯高於不治療組小鼠，顯示體內骨質疏鬆症現象減緩。另外在注射部位方面，我們也發現了雖然差異不大，但是直接將幹細胞注射進入小鼠脛骨骨髓腔，比注射在小鼠薦尾靜脈治療成效來的好。最後探討注射物質對治療成效差異，經過注射PRF+ BMSC治療的小鼠骨骼比那些單純只使用PRF或骨髓幹細胞的小鼠骨密度更加緊密。所以本研究結果證實使用PRF及BMSC可以達成骨質疏鬆症的治療效果。

Stem cells are a kind of undifferentiated cells which may differentiate into other cell types. And they can also divide indefinitely into stem cell line, but may become cancer cells at about the tenth generation. We collect the bone marrow stem cells (BMSCs) and differentiate BMSCs into osteoblast in vitro after cell sorting by flow cytometer. Platelet-rich fibrin (PRF) is a natural source of growth factors, which contains a large amount of growth factors that can facilitate bone regeneration, Such as: TGFb-1 (Transforming growth factor) , PDGF (platelet-derived growth factor) , VEGF (vascular endothelial growth factor) and Matrix proteins (thrombospondin-1, fibronectin, vitronectin). The growth factors secreted from PRF will cause cell transformation by promoting cell proliferation、matrix formation、bone formation and synthesis of collagen. We co-cultured mouse bone marrow stem cells with platelet-rich fibrin releasate (PRFr), to investigate whether the release would improve stem cell proliferation and promote cell differentiation or not. The results show that proliferation rate of the bone marrow stem cells which co-cultured with releasate were more rapid, and cell viability was much higher. Therefore, we deduced that platelet-rich fiber release solution have significant assistance on the growth and differentiation of bone marrow stem cells. To evaluate therapeutic efficacy of cell-based therapy in osteoporosis: a bone marrow stem cells (BMSCs) and platelet-rich fibrin releasate (PRFr) were used for osteoporosis treatment. An osteoporosis was established with a mouse model by ovariectomy (OVX). Transplantation of BMSCs, PRFr and BMSCs + PRFr into OVX mice for investigating the therapeutic potential for bone regeneration and recovered bone mass loss. OVX or sham operations were performed on virgin ICR mice at 16-weeks old, which were randomLy divided into three parts: Non-surgical, SHAM, and OVX. 6 mice in Non-surgical group was no surgery, 6 mice in SHAM group was subjected to sham surgery, and 30 mice in OVX group mice will accept four different treatments (1)Control group,non injection (2)BMSC-I group, injected BMSCs 3x105 cells/0.6mL PBS once a week for one weeks ; (3)PRF-I group, injected PRFr 0.6mL once a week for one weeks ; (4)BMSCs + PRF-I group, injected BMSCs 3x105 cells combine 0.6 mL PRFr) once a week for one weeks. (5)BMSC-IV group, injected BMSCs 1.2x106 BMSCs/0.6mL PBS once a week for four weeks; (6)PRF-IV group, injected 2.4mL PRFr once a week for four weeks; (7)BMSCs + PRF-IV group, injected 1.2x106 BMSCs / 2.4mL PRFr once a week for four weeks; (8)BMSCs + PRF-IV-i group, intra bone injected 1.2x106 BMSCs/ 2.4 mL PRFr once a week for four weeks. At 8 weeks after implantation, bone mass and its turnover were analyzed by micro CT, We use scanned the left tibia portion of the growth plate at the proximal 0.5-1.5mm, and analyzed bone mineral density, the percentage of bone tissue, trabecular number, trabecular thickness and trabecular separation. Then sacrifice to analyze their efficacy by histomorphometry. A statistically significant difference between the experimental and control groups was observed. BMSCs + PRFr transplants were shown effective in restoring bone mineral density. These findings indicated that the mixture of BMSC and PRF releasate could potentially be an effective agent in the treatment for osteoporosis