足底軟組織結(jié)構(gòu)體外構(gòu)建的初步研究
發(fā)布時(shí)間：2018-09-11 09:10
【摘要】： 足底軟組織創(chuàng)傷在臨床上比較常見,盡管創(chuàng)傷外科修復(fù)重建足底軟組織缺損的術(shù)式比較成熟,但是對(duì)術(shù)后功能的恢復(fù)仍不滿意。在足底軟組織缺損修復(fù)重建中,多選用小腿部皮瓣轉(zhuǎn)移修復(fù)。由于該皮瓣缺乏皮膚與深筋膜之間的縱行膠原纖維束,修復(fù)后局部軟組織穩(wěn)定性差,使得患者康復(fù)后站立不穩(wěn),行走打滑�；謴�(fù)足底軟組織特有的功能,尤其是穩(wěn)定性,是臨床難以解決的問題。 組織工程學(xué)是應(yīng)用生命科學(xué)和工程學(xué)原理與方法,研究、開發(fā)用于修復(fù)、增進(jìn)或改善人體各種組織和器官損傷后的功能和形態(tài)的一門學(xué)科。它改變了傳統(tǒng)“以創(chuàng)傷修復(fù)創(chuàng)傷”的治療模式,為最終實(shí)現(xiàn)無損傷修復(fù)創(chuàng)傷開辟了新途徑。本研究利用組織工程學(xué)理論與技術(shù)探討修復(fù)足底軟組織創(chuàng)傷,以解決局部軟組織穩(wěn)定性問題。 足底皮下軟組織結(jié)構(gòu)中主要有成纖維細(xì)胞、脂肪細(xì)胞以及少量未分化的間充質(zhì)細(xì)胞等。其中成纖維細(xì)胞數(shù)量較多,也是產(chǎn)生膠原纖維、彈性纖維和網(wǎng)狀纖維的主要細(xì)胞。這些纖維中膠原纖維數(shù)量最多,而Ⅰ型膠原纖維又占多數(shù)。有資料顯示,膠原纖維的集束還可以進(jìn)行調(diào)節(jié)。因此在體外構(gòu)建足底軟組織過程中成纖維細(xì)胞的活性以及Ⅰ型膠原蛋白分泌量和纖維束的方向就顯得至關(guān)重要。大量的研究資料表明,PTGF-BB、TGF-β1、IL-13等細(xì)胞因子對(duì)成纖維細(xì)胞具趨化作用、促進(jìn)增殖和膠原蛋白的分泌,因此本實(shí)驗(yàn)選用成纖維細(xì)胞作為種子細(xì)胞。為使成纖維細(xì)胞高分泌Ⅰ型膠原蛋白,實(shí)驗(yàn)初步選用PTGF-BB、TGF-β1、IL-13三個(gè)細(xì)胞因子作為研究對(duì)象。 組織工程的另一個(gè)重要研究方向是支架材料,理想的生物支架材料應(yīng)滿足：模仿天然細(xì)胞外基質(zhì)的結(jié)構(gòu)和生物學(xué)功能；有良好的生物相容性使細(xì)胞可粘附和增殖；降解速率與組織再生的速率相匹配；適當(dāng)?shù)臋C(jī)械性能支撐細(xì)胞生長；具有較好的顯微結(jié)構(gòu),如適合的孔隙大小、高孔隙率和孔的形態(tài)；特定的三維外形；高表面積和合適的表面理化性質(zhì)。天然細(xì)胞外基質(zhì)的膠原蛋白纖維尺寸為50-500 nm,因此納米尺度支架材料能最大程度地模仿體內(nèi)細(xì)胞外基質(zhì)的特點(diǎn)。目前靜電紡絲是惟一能直接、連續(xù)制備聚合物納米纖維的方法。所以本實(shí)驗(yàn)采用靜電紡絲法制作聚己內(nèi)酯/明膠(PCL/gelatin)復(fù)合納米纖維作為支架材料,它不僅具有表面積大、孔徑小、孔隙率高、較好的均一性,直徑與體內(nèi)細(xì)胞外基質(zhì)中膠原纖維相近,能很好的模仿細(xì)胞外基質(zhì)的天然結(jié)構(gòu)。而且還避免了單用PCL導(dǎo)致降解慢,生物相容性差,和單用明膠導(dǎo)致降解太快的問題。通過調(diào)節(jié)PCL、gelatin之間不同的混合比例,從而得到我們需要的降解率和生物相容性。 本實(shí)驗(yàn)采用出生2天的SD大鼠背部皮膚做原代培養(yǎng),以獲得種子細(xì)胞。應(yīng)用不同的細(xì)胞因子作用于種子細(xì)胞,以研究這些細(xì)胞因子對(duì)成纖維細(xì)胞的增殖及分泌Ⅰ型膠原蛋白的影響程度。將種子細(xì)胞與PCL/gelatin納米支架材料復(fù)合通過觀察種子細(xì)胞在材料表面的生長情況和檢測(cè)培養(yǎng)液中Ⅰ型膠原蛋白的濃度,初步的評(píng)價(jià)PCL/gelatin納米支架材料的生物相容性和應(yīng)用到本課題的可行性以及為以后在大鼠體內(nèi)實(shí)驗(yàn)和臨床應(yīng)用提供實(shí)驗(yàn)參考。實(shí)驗(yàn)主要有以下兩個(gè)部分： 目的：選用不同濃度的血小板源性生長因子BB (PDGF-BB)、白介素13(IL-13)、轉(zhuǎn)化生長因子β1 (TGF-β1)在體外誘導(dǎo)真皮成纖維細(xì)胞,以研究不同的細(xì)胞因子對(duì)成纖維細(xì)胞的增殖及分泌Ⅰ型膠原蛋白的影響程度。 方法：選用出生2d的SD大鼠背部皮膚進(jìn)行真皮成纖維細(xì)胞原代培養(yǎng),采用免疫熒光技術(shù)對(duì)其進(jìn)行純度鑒定。實(shí)驗(yàn)分為四組,分別是PDGF-BB (30ng/ml)組,IL-13(100ng/m)組,TGF-β1 (10ng/ml)組和不加任何處理因素的陰性對(duì)照組,用MTT法、ELISA檢測(cè)在24h、48h、72h時(shí)的真皮成纖維細(xì)胞的增殖情況及培養(yǎng)液中Ⅰ型膠原蛋白的濃度。用RT-PCR法檢測(cè)各組中真皮成纖維細(xì)胞Ⅰ型膠原蛋白基因的表達(dá)情況。 結(jié)果：原代培養(yǎng)的真皮成纖維細(xì)胞的純度達(dá)90%以上。PDGF-BB、IL-13、TGF-β1均能促進(jìn)真皮成纖維細(xì)胞增殖和Ⅰ型膠原蛋白分泌,在48h,72h時(shí)PDGF-BB組的培養(yǎng)液中Ⅰ型膠原蛋白濃度顯著高于IL-13組,TGF-β1組及陰性對(duì)照組(P0.05)。初步RT-PCR分析顯示PDGF-BB組Ⅰ型膠原蛋白基因表達(dá)相對(duì)多于其它各組及對(duì)照組。 結(jié)論：PDGF-BB、IL-13、TGF-β1均能促進(jìn)真皮成纖維細(xì)胞增殖和分泌Ⅰ型膠原蛋白,其中濃度為30ng/ml的PDGF-BB的作用更為顯著。 目的：通過觀察真皮成纖維細(xì)胞在聚己內(nèi)酯/明膠(PCL/gelatin)復(fù)合納米支架材料上生長情況以及檢測(cè)培養(yǎng)液中的Ⅰ型膠原蛋白的濃度從而初步的評(píng)價(jià)PCL/gelatin復(fù)合納米纖維的生物相容性。 方法：采用靜電紡絲法制備PCL/gelatin復(fù)合納米纖維,將原代培養(yǎng)的真皮成纖維細(xì)胞接種到材料的表面,用掃描電鏡觀察真皮成纖維細(xì)胞在材料表面的生長情況。實(shí)驗(yàn)分為3組：支架材料組、PDGF-BB組、陰性對(duì)照組,應(yīng)用ELISA法檢測(cè)細(xì)胞液中Ⅰ型膠原蛋白的濃度,采用重復(fù)測(cè)量方差分析和LSD多重比較法比較各組培養(yǎng)液中的Ⅰ型膠原蛋白濃度。 結(jié)果：PCL、gelatin混合溶液用靜電紡技術(shù)制備出的PCL/gelatin納米纖維呈現(xiàn)相互連通的三維網(wǎng)絡(luò)狀結(jié)構(gòu)；纖維的直徑與分布范圍較均勻,其直徑為700nm左右。真皮成纖維細(xì)胞在PCL/gelatin納米支架材料表面貼附牢固,具有良好的生長形態(tài),呈現(xiàn)長梭形或者多角形,伸展充分。細(xì)胞與纖維之間、細(xì)胞與細(xì)胞之間均有較多樹枝狀的突起相互交連,還可觀察到細(xì)胞向纖維材料內(nèi)部長入的現(xiàn)象。培養(yǎng)液中Ⅰ型膠原蛋白的檢測(cè)結(jié)果統(tǒng)計(jì)顯示,培養(yǎng)24h后各組間培養(yǎng)液中的Ⅰ型膠原蛋白的濃度無顯著差異(P＞0.05)。在培養(yǎng)48h,72h后,PDGF-BB組培養(yǎng)液中的Ⅰ型膠原蛋白的濃度顯著高于支架材料組和對(duì)照組(P0.05)。支架材料組與對(duì)照組在不同時(shí)間培養(yǎng)液中Ⅰ型膠原蛋白的濃度均無顯著性差異(P0.05)。 結(jié)論：PCL/gelatin復(fù)合納米材料具有較好的生物相容性,可以作為足底皮下軟組織構(gòu)建的潛在支架材料。
[Abstract]:Trauma of plantar soft tissue is common in clinic. Although the surgical method of repairing and reconstructing plantar soft tissue defect is mature, the functional recovery is still unsatisfactory. The instability of the local soft tissue after the repair of the fibrous tract makes the patient stand unstable and walk slippery after recovery. It is difficult to restore the unique function of the plantar soft tissue, especially the stability.
Tissue engineering is a subject that applies the principles and methods of life science and engineering to study and develop for repairing, enhancing or improving the function and morphology of human tissues and organs after injury. To study the application of tissue engineering theory and technology in repairing plantar soft tissue trauma and to solve the problem of local soft tissue stability.
There are mainly fibroblasts, adipocytes and a few undifferentiated mesenchymal cells in the subcutaneous tissue of plantar. Among them, fibroblasts are the main cells producing collagen fibers, elastic fibers and reticular fibers. The results indicate that the aggregation of collagen fibers can also be regulated. Therefore, the activity of fibroblasts and the secretion of collagen type I and the direction of fibrous bundles are very important in the process of plantar soft tissue construction in vitro. In order to make fibroblasts secrete type I collagen, three cytokines, PTGF-BB, TGF-beta 1 and IL-13, were selected as the research objects.
Another important research direction of tissue engineering is scaffold material, which should be satisfied with: mimic the structure and biological function of natural extracellular matrix; have good biocompatibility to enable cells to adhere and proliferate; degradation rate matches the rate of tissue regeneration; appropriate mechanical properties to support cell growth. It has good microstructure, such as suitable pore size, high porosity and pore morphology, specific three-dimensional shape, high surface area and suitable surface physical and chemical properties. At present, electrospinning is the only direct and continuous method to prepare polymer nanofibers. In this experiment, polycaprolactone/gelatin (PCL/gelatin) composite nanofibers were prepared by electrospinning as scaffolds. It has not only large surface area, small pore size, high porosity, good uniformity, diameter and in vivo and in vitro matrix gum. By adjusting the mixing ratio of PCL and gelatin, we can get the degradation rate and biocompatibility we need.
Seed cells were obtained from the back skin of two-day-old SD rats by primary culture. Seed cells were treated with different cytokines to study the effects of these cytokines on the proliferation and secretion of type I collagen in fibroblasts. The growth of seed cells on the surface of the material and the concentration of collagen type I in the culture medium were measured. The biocompatibility of PCL/gelatin nano-scaffolds and the feasibility of its application in this study were preliminarily evaluated. The experiment provided experimental reference for the future experiment and clinical application in rats.
AIM: To investigate the effects of platelet-derived growth factor BB (PDGF-BB), interleukin-13 (IL-13) and transforming growth factor-beta 1 (TGF-beta 1) on the proliferation and type I collagen secretion of fibroblasts in vitro.
METHODS: Dermal fibroblasts were cultured on the dorsal skin of SD rats born 2 days after birth and purified by immunofluorescence technique. The experiment was divided into four groups: PDGF-BB (30ng/ml), IL-13 (100ng/m), TGF-beta 1 (10ng/ml) and negative control group without any treatment factors. MTT and ELISA were used to detect the purity of dermal fibroblasts at 24h and 48h. The proliferation of dermal fibroblasts at 72 h and the concentration of collagen type I in culture medium were detected by RT-PCR.
Results: The purity of primary cultured dermal fibroblasts was over 90%. PDGF-BB, IL-13, TGF-beta 1 could promote the proliferation of dermal fibroblasts and the secretion of type I collagen. The concentration of type I collagen in culture medium of PDGF-BB group was significantly higher than that of IL-13 group at 48h and 72h, TGF-beta 1 group and negative control group (P 0.05). Preliminary RT-PCR analysis showed that P The expression of type I collagen gene in group DGF-BB was more than that in other groups and control groups.
Conclusion: PDGF-BB, IL-13 and TGF-beta 1 can promote the proliferation and secretion of type I collagen in dermal fibroblasts, and the effect of PDGF-BB at the concentration of 30 ng/ml is more significant.
AIM: To evaluate the biocompatibility of PCL/gelatin composite nanofibers by observing the growth of dermal fibroblasts on polycaprolactone/gelatin (PCL/gelatin) composite nanoscaffolds and detecting the concentration of collagen type I in culture medium.
METHODS: PCL/gelatin composite nanofibers were prepared by electrospinning method. The primary cultured dermal fibroblasts were seeded onto the surface of the material and the growth of the dermal fibroblasts on the surface of the material was observed by scanning electron microscopy. The concentration of type I collagen in culture medium was compared by repeated analysis of variance and LSD multiple comparison.
Results: PCL/gelatin nanofibers prepared by electrospinning with PCL and gelatin mixed solution showed a three-dimensional interconnected network structure; the diameter and distribution of the fibers were uniform, and the diameter of the fibers was about 700 nm. Dermal fibroblasts adhered firmly to the surface of PCL/gelatin nano-scaffolds with good growth morphology. There are many dendritic processes between cells and fibers, and there are many dendritic processes between cells and cells. The phenomena of cells growing into fibrous materials can also be observed. The concentration of type I collagen in PDGF-BB group was significantly higher than that in scaffold material group and control group at 48h and 72h after culture (P > 0.05).
CONCLUSION: PCL/gelatin composite nanomaterials have good biocompatibility and can be used as potential scaffolds for plantar subcutaneous soft tissue construction.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R322
本文編號(hào)：2236257
[Abstract]:Trauma of plantar soft tissue is common in clinic. Although the surgical method of repairing and reconstructing plantar soft tissue defect is mature, the functional recovery is still unsatisfactory. The instability of the local soft tissue after the repair of the fibrous tract makes the patient stand unstable and walk slippery after recovery. It is difficult to restore the unique function of the plantar soft tissue, especially the stability.
Tissue engineering is a subject that applies the principles and methods of life science and engineering to study and develop for repairing, enhancing or improving the function and morphology of human tissues and organs after injury. To study the application of tissue engineering theory and technology in repairing plantar soft tissue trauma and to solve the problem of local soft tissue stability.
There are mainly fibroblasts, adipocytes and a few undifferentiated mesenchymal cells in the subcutaneous tissue of plantar. Among them, fibroblasts are the main cells producing collagen fibers, elastic fibers and reticular fibers. The results indicate that the aggregation of collagen fibers can also be regulated. Therefore, the activity of fibroblasts and the secretion of collagen type I and the direction of fibrous bundles are very important in the process of plantar soft tissue construction in vitro. In order to make fibroblasts secrete type I collagen, three cytokines, PTGF-BB, TGF-beta 1 and IL-13, were selected as the research objects.
Another important research direction of tissue engineering is scaffold material, which should be satisfied with: mimic the structure and biological function of natural extracellular matrix; have good biocompatibility to enable cells to adhere and proliferate; degradation rate matches the rate of tissue regeneration; appropriate mechanical properties to support cell growth. It has good microstructure, such as suitable pore size, high porosity and pore morphology, specific three-dimensional shape, high surface area and suitable surface physical and chemical properties. At present, electrospinning is the only direct and continuous method to prepare polymer nanofibers. In this experiment, polycaprolactone/gelatin (PCL/gelatin) composite nanofibers were prepared by electrospinning as scaffolds. It has not only large surface area, small pore size, high porosity, good uniformity, diameter and in vivo and in vitro matrix gum. By adjusting the mixing ratio of PCL and gelatin, we can get the degradation rate and biocompatibility we need.
Seed cells were obtained from the back skin of two-day-old SD rats by primary culture. Seed cells were treated with different cytokines to study the effects of these cytokines on the proliferation and secretion of type I collagen in fibroblasts. The growth of seed cells on the surface of the material and the concentration of collagen type I in the culture medium were measured. The biocompatibility of PCL/gelatin nano-scaffolds and the feasibility of its application in this study were preliminarily evaluated. The experiment provided experimental reference for the future experiment and clinical application in rats.
AIM: To investigate the effects of platelet-derived growth factor BB (PDGF-BB), interleukin-13 (IL-13) and transforming growth factor-beta 1 (TGF-beta 1) on the proliferation and type I collagen secretion of fibroblasts in vitro.
METHODS: Dermal fibroblasts were cultured on the dorsal skin of SD rats born 2 days after birth and purified by immunofluorescence technique. The experiment was divided into four groups: PDGF-BB (30ng/ml), IL-13 (100ng/m), TGF-beta 1 (10ng/ml) and negative control group without any treatment factors. MTT and ELISA were used to detect the purity of dermal fibroblasts at 24h and 48h. The proliferation of dermal fibroblasts at 72 h and the concentration of collagen type I in culture medium were detected by RT-PCR.
Results: The purity of primary cultured dermal fibroblasts was over 90%. PDGF-BB, IL-13, TGF-beta 1 could promote the proliferation of dermal fibroblasts and the secretion of type I collagen. The concentration of type I collagen in culture medium of PDGF-BB group was significantly higher than that of IL-13 group at 48h and 72h, TGF-beta 1 group and negative control group (P 0.05). Preliminary RT-PCR analysis showed that P The expression of type I collagen gene in group DGF-BB was more than that in other groups and control groups.
Conclusion: PDGF-BB, IL-13 and TGF-beta 1 can promote the proliferation and secretion of type I collagen in dermal fibroblasts, and the effect of PDGF-BB at the concentration of 30 ng/ml is more significant.
AIM: To evaluate the biocompatibility of PCL/gelatin composite nanofibers by observing the growth of dermal fibroblasts on polycaprolactone/gelatin (PCL/gelatin) composite nanoscaffolds and detecting the concentration of collagen type I in culture medium.
METHODS: PCL/gelatin composite nanofibers were prepared by electrospinning method. The primary cultured dermal fibroblasts were seeded onto the surface of the material and the growth of the dermal fibroblasts on the surface of the material was observed by scanning electron microscopy. The concentration of type I collagen in culture medium was compared by repeated analysis of variance and LSD multiple comparison.
Results: PCL/gelatin nanofibers prepared by electrospinning with PCL and gelatin mixed solution showed a three-dimensional interconnected network structure; the diameter and distribution of the fibers were uniform, and the diameter of the fibers was about 700 nm. Dermal fibroblasts adhered firmly to the surface of PCL/gelatin nano-scaffolds with good growth morphology. There are many dendritic processes between cells and fibers, and there are many dendritic processes between cells and cells. The phenomena of cells growing into fibrous materials can also be observed. The concentration of type I collagen in PDGF-BB group was significantly higher than that in scaffold material group and control group at 48h and 72h after culture (P > 0.05).
CONCLUSION: PCL/gelatin composite nanomaterials have good biocompatibility and can be used as potential scaffolds for plantar subcutaneous soft tissue construction.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R322
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