探討利用玻尿酸微環境因子引導脂肪幹細胞軟骨分化 以應用於關節軟骨組織工程

The study of applying hyaluronan microenvironmental factor in chondrogenic induction of adipose derived stem cells for articular cartilage tissue engineering

吳順成(Shun-Cheng Wu)

指導教授 ： 王國照

高雄醫學大學/醫學院/醫學研究所/博士(2010年)

https://doi.org/10.6832/KMU.2010.00019

摘要

由於關節軟骨自我修復的能力相當有限，因此如何修復關節軟骨成為肌肉骨骼醫學上的重大挑戰之一。以細胞為基礎的組織工程方式進行修補，被認為相較於目前的修補方式而言，是擁有較多優點的修補方法。組織工程包含三個主要之組成要素: 1.細胞(Cells)。2.生物材料(Biomaterials)。3. 環境因子(Environmental factors)。研究人員在修補關節軟骨上所致力發展方法以解決下列之問題: 一.如何取得足量之未分化的間質幹細胞或已分化的軟骨細胞。二.如何使用適當的生物材料或生長因子以期維持移植之軟骨細胞之表型，並在移植處生成透明軟骨基質的生成。本論文在修補關節軟骨上所提出之策略如下:為解決關節軟骨缺損之修復問題提出解決之方法，1.)移植人類脂肪幹細胞(Human adipose derived stem cells; hADSCs)以解決軟骨細胞來源缺少之問題。2.)應用玻尿酸(Hyaluronan; HA)做為微環境因子，以起始及促進脂肪幹細胞進行軟骨分化做為關節軟骨組織工程之用。
本研究包含兩部份: 第一部份探討富含玻尿酸微環境對於人類脂肪幹細胞軟骨分化的促進作用。我們以玻尿酸覆蓋之培養皿及以玻尿酸表面改質之Poly(DL-lactic-co-glycolic acid) (PLGA)支架做為玻尿酸微環境。結果顯示:培養人類脂肪幹細胞於玻尿酸表面覆蓋培養盤(0.005-0.5 mg/cm2)之中，細胞聚合及軟骨分化基因(SOX-9, Collagen type II, Aggrecan)的基因表現量於24小時有增加之現象，且聚葡萄糖胺含量於第9天時也有顯著性增加。以玻尿酸進行表面改質的PLGA支架(HA/PLGA支架)相較於PLGA支架而言，並不會影響人類脂肪幹細胞的細胞貼附能力及細胞存活能力。相較於培養於PLGA支架而言，人類脂肪幹細胞於HA/PLGA支架，軟骨分化軟骨分化基因之表現量於培養的第1到5天有顯著性增加，且纖維軟骨基因及軟骨肥大基因則顯著性下降。且培養4週之後觀察關節軟骨組織生成，培養於HA/PLGA支架之人類脂肪幹細胞生成較多的聚葡萄糖胺及第二型膠原蛋白。我們的結果顯示玻尿酸微環境可引導人類脂肪幹細胞軟骨分化且生成透明軟骨基質而此特性可應用於關節軟骨組織工程。
第二部份，基於第一部份的發現我們更進一步去探討玻尿酸微環境啟動及促進人類脂肪幹細胞之分子機制。在這部份，我們假設人類脂肪幹細胞及玻尿酸之間的niche interaction是促進軟骨化之原因，且此效應主要透過人類脂肪幹細胞上之玻尿酸受體CD44。玻尿酸及CD44結合(HA-CD44)的niche interaction之分子機制，我們利用培養人類脂肪幹細胞於玻尿酸覆蓋之培養皿上做探討，人類脂肪幹細胞之關節軟骨細胞外間質合成，則是將人類脂肪幹細胞及玻尿酸一起培養於三維纖維蛋白水膠做測試。結果顯示:99.9%之人類脂肪幹細胞表現CD44在細胞表面。人類脂肪幹細胞於玻尿酸覆蓋之培養皿上時發現hyaluronidase-1(Hyal-1)之表現量上升，顯示了CD44之細胞內訊息傳遞已被HA-CD44所促進，軟骨分化基因(SOX-9, Collagen type II, Aggrecan)的基因表現量於1到5天也有增加之現象。此外抑制HA-CD44可完全抑制Hyal-1基因且也減少軟骨化基因之表現量。人類脂肪幹細胞培養於3維纖維蛋白水膠之細胞存活並沒有受到外加玻尿酸而改變。較高之Hyal-1及軟骨分化基因表現量，顯示人類脂肪幹細胞培養混合玻尿酸之水膠(HA/fibrin hydrogel)之中，HA-CD44啟動了人類脂肪幹細胞之細胞內訊息傳遞及促進軟骨分化。較高量之聚葡萄糖胺(sGAG)及細胞內膠原蛋白(Total intracellular collagen)合成也顯示了在HA/fibrin hydrogel中較強烈之軟骨基質合成。從免疫細胞染色之中發現，相較於沒有混合玻尿酸之纖維蛋白水膠，在HA/fibrin hydrogel中第二型膠原蛋白被明顯染色，第十型膠原蛋白則較少被染色。我們的結果顯示HA-CD44是玻尿酸微環境所引導之人類脂肪幹細胞軟骨分化及軟骨細胞外間質合成之主要因素。這些結果富含玻尿酸niche所引導及促進人類脂肪幹細胞之軟骨分化主要是由於CD44所造成之訊息傳遞且再促進軟骨基質之合成。促進HA-CD44訊息傳遞或許可以應用於以脂肪幹細胞為基礎之關節軟骨重建。
綜合上述之結果，玻尿酸可做為一個可起始及促進人類脂肪幹細胞軟骨分化之微環境因子。此外玻尿酸也可以促進人類脂肪幹細胞生成透明軟骨基質。而玻尿酸之促進軟骨分化及透明軟骨基質合成之原因主要是透過在人類脂肪幹細胞上之HA-CD44起使訊息傳遞。這些發現顯示玻尿酸微環境因子或許可應用於較有效之以脂肪幹細胞為基礎之關節軟骨組織工程。

關鍵字

關節軟骨組織工程 ； 脂肪幹細胞 ； 玻尿酸 ； 微環境 ； 軟骨分化

並列摘要

Due to the poor intrinsic ability of articular cartilage for repair, injuries to this tissue are one of the most challenging issues of musculoskeletal medicine. The recent stem cell based tissue engineering has offers many advantages over current techniques in the repair of damaged cartilage. Tissue engineering consists of three major components such as cells, biomaterials and environmental factors. Researchers are developing methods for reconstruction of the damaged articular cartilage in order to resolve the problems as follows: 1) How to get the appropriate quantity of the undedifferentiated mesenchymal stem cells or differentiated chondrocytes. 2) How to use the proper biomaterials or growth factors in order to maintain the phenotype of the transplanted chondrocytes and maintain the production of hyaline cartilage matrix in the repair site. The strategies to approach the articular cartilage tissue engineering in this thesis are as following: 1.) Transplantation of human adipose derived stem cells (hADSCs) in order to resolve the shortage of the source of chondrocytes. 2.) Using hyaluronan (HA) as a microenvironmental factor (niche) for initiating and promoting chondrogenesis of ADSCs for articular cartilage tissue engineering.
This study consists of two parts: In the first part we focused on the enhancing effect of HA-enriched microenvironment on chondrogenesis of hADSCs. The HA-coated wells and HA-modified poly-(lactic-co-glycolic acid) (HA/PLGA) scaffolds were used as the HA-enriched microenvironment. The results showed that hADSCs cultured in HA-coated wells (0.005-0.5 mg/cm2) showed enhancing aggregation and chondrogenic mRNA expressions (SOX-9, collagen type II, and aggrecan) after 24 hrs, and increasing sulfated glycosaminoglycan (sGAG) content after 9 days of culture. The HA/PLGA scaffolds did not change the cell adherence and viability of hADSCs. The mRNA expressions of chondrogenic marker genes were significantly enhanced in hADSCs cultured in HA/PLGA rather than those cultured in the PLGA scaffold after 1, 3, and 5 days of culture. The hADSCs cultured in HA/PLGA produced higher levels of sGAG and collagen type II, compared to those in the PLGA scaffold after 4 weeks of cultures. Our results suggest that HA-enriched microenvironment induces chondrogenesis and promotes hyaline cartilaginous matrix formation in hADSCs, which may be beneficial in articular cartilage tissue engineering.
In part II, based on the findings of part I we further investigated the molecular mechanism of the enhancing effect of HA-enriched microenvironment in initiating and promoting chondrogenesis of hADSCs. In this part, we further hypothesize that the niche interaction of hADSCs and HA contributes to chondrogenesis and is mainly through CD44, the cell surface receptor of HA, in hADSCs. The molecular mechanism of niche interaction between HA and CD44 binding (HA-CD44) was tested by hADSCs cultured in HA-coated wells, and the cartilaginous matrix formation of hADSCs was further tested by culturing hADSCs in a three-dimensional (3D) fibrin hydrogel mixing with HA. The result showed that 99.9% of hADSCs possess CD44 on cell surface. Enhancement of hyaluronidase-1 (Hyal-1) expression of hADSCs cultured on HA-coated wells indicated the intra-cellular signaling of CD44 has been enhanced by HA-CD44. Chondrogenic marker gene expressions (SOX-9, collagen type II and aggrecan) in hADSCs were also up-regulated 1 to 5 days after cultured on HA-coated wells. Furthermore, blocking the HA-CD44 inhibited Hyal-1 gene expression and reduced chondrogenic marker gene expressions. The result from the hADSCs cultured in 3D fibrin hydrogel showed that the cell survival did not altered by HA addition. Higher levels of Hyal-1 and chondrogenic marker genes expressions found in hADSCs cultured in hydrogel with HA (HA/fibrin hydrogel) confirmed the HA-CD44 initiating intra-cellular signaling and HA enhanced chondrogenesis, respectively. Higher sulfated glycosaminoglycan (sGAG) and total collagen levels were also found in the HA/fibrin hydrogel group indicated more pronounced cartilaginous matrix formation. The result from immunocytochemistry showed that collagen type II was obviously stained, but collagen type X was rarely stained in HA/fibrin hydrogel rather than in fibrin hydrogel without HA. Our results indicated that CD44-HA interaction is the main contribution to the HA microenvironment induced chondrogenesis of hADSCs and cartilaginous matrix formation. This finding suggests that HA-enriched niche initiating and enhancing chondrogenesis of hADSCs is mainly attributed to CD44 signaling, and subsequently increases cartilage matrix formation. Enhancement of HA-CD44 signaling may be applied to the ADSC-based cartilage regeneration.
Taken together, with results from part I and II studies, HA as a microenvironmental factor can both initiate and enhance chondrogenic differentiation of hADSCs. Other than that, it can also promote the hyaline cartilaginous matrix formation of hADSCs. These enhancing effects of HA on chondrogenic differentiation and hyaline cartilaginous matrix formation is mainly through the niche interaction via HA-CD44 adhesion mediated signaling in hADSCs. These findings indicate that it may be applied for more effective ADSC-based articular cartilage tissue engineering.

並列關鍵字

articular cartilage tissue engineering ； adipose derived stem cells ； hyaluronan ； microenvironment ； chondrogenesis

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