Fetal tissue engineering: chest wall reconstruction

Background/Purpose: This study was aimed at applying fetal tissue engineering to chest wall reconstruction.Methods: Fetal lambs underwent harvest of elastic and hyaline cartilage specimens. Once expanded in vitro, fetal chondrocytes were seeded onto synthetic scaffolds, which then were placed in a bioreactor. After birth, fetal cartilage constructs (n = 10) were implanted in autologous fashion into the ribs of all lambs (n = 6) along with identical, but acellular scaffolds, as controls (n = 6). Engineered and acellular specimens were harvested for analysis at 4 to 12 weeks postimplantation. Standard histology and matrix-specific staining were performed both before implantation and after harvest on all constructs.Results: Regardless of the source of chondrocytes, all fetal constructs resembled hyaline cartilage, both grossly and histologically, in vitro. In vivo, engineered implants retained hyaline characteristics for up to 10 weeks after implantation but remodeled into fibrocartilage by 12 weeks postoperatively. Mononuclear inflammatory infiltrates surrounding residual PGA/PLLA polymer fibers were noted in all specimens but most prominently in the acellular controls.Conclusions: Engineered fetal cartilage can provide structural replacement for at least up to 10 weeks after autologous, postnatal implantation in the chest wall. Fetal tissue engineering may prove useful for the treatment of severe congenital chest wall defects at birth.     

Fetal tissue engineering: in utero tracheal augmentation in an ovine model

Background/Purpose: This study was aimed at comparing fetal tissue engineering with autologous free grafting in an ovine model of in utero tracheal repair. Methods: Chondrocytes were isolated from both elastic and hyaline cartilage specimens harvested from fetal lambs and expanded in vitro. Cells were seeded dynamically onto biodegradable scaffolds, which then were maintained in a rotating bioreactor for 6 to 8 weeks. Constructs subsequently were implanted into fetal tracheas (n = 15), in a heterologous fashion (group I). In group II, fetuses (n = 5) received autologous free grafts of elastic cartilage harvested from the ear as tracheal implants. In vivo specimens were harvested for histologic analysis at different time-points postimplantation. Results: In the 12 of 15 surviving fetuses of group I, all constructs were found to resemble normal hyaline cartilage, engraft well despite their heterologous origin, and display time-dependent epithelialization derived from the native trachea. All autologous free grafts were engrafted and epithelialized at birth, retaining histologic characteristics of elastic cartilage, but were more deformed than engineered constructs. Of the lambs allowed to reach term, 5 of 5 in the engineered group and 4 of 5 in the free graft group could breathe spontaneously. Conclusions: (1) Tissue-engineered cartilage, as well as autologous free grafts, can be implanted successfully into the fetal trachea, resulting in engraftment and function. (2) Engineered cartilage provides enhanced structural support after implantation into the fetal trachea when compared with free grafts. Prenatal tracheoplasty may prove useful for the treatment of severe congenital tracheal malformations. J Pediatr Surg 37:1000-1006.     

组织工程骨软骨复合物的构建与形态学观察

目的探讨采用组织工程技术构建骨软骨复合物的可行性。方法将骨髓基质细胞(BMSCs)成诱导软骨后接种于快速成形的三维支架材料聚乳酸／聚羟乙酸共聚物(PLGA)构建组织工程软骨，经成骨诱导的BMSCs接种于聚乳酸／聚羟乙酸共聚物／磷酸三钙(PLGA／TCP)构建组织工程骨，在体外分别培养2周后，将两种工程化组织及两者以无损伤线缝合形成的组织工程骨软复合体分别植入自体股部肌袋，术后8周取材，行组织学观察。结果术后组织学观察表明。组织工程软骨在体内可形成软骨组织组织工程骨在体内可形成骨组织，两者的复合体在体内可形成骨软骨复合物。结论以骨髓基质细胞为种子细胞、以快速成形的生物降解材料为支架体外构建的组织工程骨软骨复合物，可在体内形成骨软骨组织，有望用于骨软骨缺损的修复。     

Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: implantation method and short-term results

OBJECTIVE: To compare four different implantation modalities for the repair of superficial osteochondral defects in a caprine model using autologous, scaffold-free, engineered cartilage constructs, and to describe the short-term outcome of successfully implanted constructs. METHODS: Scaffold-free, autologous cartilage constructs were implanted within superficial osteochondral defects created in the stifle joints of nine adult goats. The implants were distributed between four 6-mm-diameter superficial osteochondral defects created in the trochlea femoris and secured in the defect using a covering periosteal flap (PF) alone or in combination with adhesives (platelet-rich plasma (PRP) or fibrin), or using PRP alone. Eight weeks after implantation surgery, the animals were killed. The defect sites were excised and subjected to macroscopic and histopathologic analyses. RESULTS: At 8 weeks, implants that had been held in place exclusively with a PF were well integrated both laterally and basally. The repair tissue manifested an architecture similar to that of hyaline articular cartilage. However, most of the implants that had been glued in place in the absence of a PF were lost during the initial 4-week phase of restricted joint movement. The use of human fibrin glue (FG) led to massive cell infiltration of the subchondral bone. CONCLUSIONS: The implantation of autologous, scaffold-free, engineered cartilage constructs might best be performed beneath a PF without the use of tissue adhesives. Successfully implanted constructs showed hyaline-like characteristics in adult goats within 2 months. Long-term animal studies and pilot clinical trials are now needed to evaluate the efficacy of this treatment strategy.     

Donor fascia in urogynaecological procedures: a canine model

OBJECTIVE: To explore the in vivo characteristics of donor fascia used in urogynaecological procedures, in a canine model. MATERIALS AND METHODS: Two experiments were conducted. In the first, donor fascia grafts were obtained from 12 dogs, the grafts freeze-dried and half were irradiated. The grafts were used for sacrocolpopexy and suburethral slings in each of five dogs. The dogs were killed at 2, 6 and 12 weeks after graft implantation, the grafts retrieved and assessed using tensilometry. In the second experiment, unirradiated sacrocolpopexy grafts were implanted in eight dogs; four grafts were placed under no tension and four under moderate tension. At 8 weeks, the grafts were retrieved and assessed by tensilometry. Measures of strength in both experiments included the ultimate tensile strength, ultimate strain and stiffness. All measures were compared using Kruskal-Wallis nonparametric tests in both studies. RESULTS: In the first experiment, a significant minority (23%) of grafts had complete loss of strength. Measures of graft strength did not vary when analysed according to donor dog, host dog, history of graft irradiation, duration of implantation or location of graft. In the second experiment, grafts placed under no tension tended to have lower tensile strength (chi2(1) = 3.125, P = 0.077), lower stiffness (chi2(1) = 3.125, P = 0.077) and lower ultimate strain (chi 2(1) = 3.182, P = 0.074). CONCLUSION: Graft irradiation as an isolated variable did not predispose grafts to failure in vivo. Biomechanical factors at the implantation site are likely to play a critical role in determining ultimate graft strength.     

利用软骨细胞膜片技术在山羊皮下构建软骨样组织的研究

目的 探讨软骨细胞膜片技术在大型哺乳动物体内构建软骨的优越性.方法 以山羊耳廓软骨细胞为种子细胞,分别利用细胞膜片技术(实验组)和细胞复合PGA/PLA支架材料的方法(对照组)构建软骨组织.体外培养4周回植到动物腹部皮下,分别于回植前、回植后4周及回植后8周时采集标本,观察比较两组软骨形成情况.结果 体外培养4周后,两组均形成一定量的软骨样组织,但对照组可见未降解的材料纤维;体内构建4周后,实验组形成成熟软骨样组织,对照组为纤维结缔组织替代,伴有大量炎性细胞浸润;体内培养8周时,实验组较4周前无明显变化,对照组体积明显缩小,仍为纤维结缔组织.结论 软骨细胞膜片技术与细胞复合PGA/PLA支架材料构建组织工程软骨方法相比,原代细胞用量少,体外培养时间短,在大动物体内能形成成熟软骨,具有更广泛的临床应用前景.     

Small-diameter blood vessels engineered with bone marrow-derived cells

OBJECTIVE: The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. SUMMARY BACKGROUND DATA: BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. METHODS: Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. RESULTS: The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. CONCLUSIONS: Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.     

Prenatal tracheal reconstruction with a hybrid amniotic mesenchymal stem cells-engineered construct derived from decellularized airway

PurposeThis study was aimed at examining an airway construct engineered from autologous amniotic mesenchymal stem cells (aMSCs) and a xenologous decellularized airway scaffold as a means for tracheal repair.MethodsFetal lambs (N = 13) with a tracheal defect were divided into 2 groups. One group (acellular, n = 6) was repaired with a decellularized leporine tracheal segment. The other group (engineered, n = 7) received an identical graft seeded with expanded/labeled autologous aMSCs. Newborns were euthanized for multiple analyses.ResultsEleven lambs survived to term, 10 of which could breathe at birth. Engineered grafts showed a significant increase in diameter in vivo (P = .04) unlike acellular grafts (P = .62), although variable stenosis was present in all implants. Engineered constructs exhibited full epithelialization, compared with none of the acellular grafts (P = .002). Engineered grafts had a significantly greater degree of increase in elastin levels after implantation than acellular implants (P = .04). No such differences were noted in collagen and glycosaminoglycan contents. Donor cells were detected in engineered grafts, which displayed a pseudostratified columnar epithelium.ConclusionsConstructs engineered from aMSCs and decellularized airway undergo enhanced remodeling and epithelialization in vivo when compared with equivalent acellular implants. Amniotic mesenchymal stem cell–engineered airways may become an alternative for perinatal airway repair.     

Modified mesh for hernia repair that is adapted to the physiology of the abdominal wall.

OBJECTIVE: To develop a new mesh for hernia repair that is adapted to the physiological forces. DESIGN: Animal experiment. SETTING: Surgical Department of the RWTH-Aachen. ANIMALS: Wistar rats MAIN OUTCOME MEASURES: Textile analysis, tensile strength, bending stiffness, histology and morphometry. RESULTS: After textile analysis of commercially available meshes in clinical use we defined the physiological forces and constructed a new mesh (Soft Hernia Mesh, SHM) based on a combination of non-absorbable polypropylene and absorbable polyglactin 910. The amount of non-absorbable material could be reduced to < 30% compared with Marlex while still guaranteeing the necessary pulling force of 16 N/cm. Improvements of the hosiery structure improved the symmetrical distribution of the retaining forces in all directions. Compared with the considerable restriction of the abdominal wall mobility by Prolene (polypropylene) and Mersilene (polyester) meshes there was no increase in the bending stiffness after the implantation of the new mesh. Histological examination showed a pronounced reduction of the inflammatory reaction in the tissues, and the collagen bundles were orientated merely around the mesh filaments instead of forming a scar plate that completely embedded the mesh. CONCLUSION: Different meshes caused specific histological reactions with changes of their mechanical properties after implantation in rodents. A new mesh with a reduced amount of polypropylene showed both less inflammation and less restriction in the mobility of the abdominal wall though it exceeded the required tensile strength of 16 N/cm.     

Adjacent tissues (cartilage, bone) affect the functional integration of engineered calf cartilage in vitro

OBJECTIVE: An in vitro model was used to test the hypothesis that culture time and adjacent tissue structure and composition affected chondrogenesis and integrative repair in engineered cartilage. METHOD: Engineered constructs made of bovine calf chondrocytes and hyaluronan benzyl ester non-woven mesh were press-fitted into adjacent tissue rings made of articular cartilage (AC), devitalized bone (DB), or vital bone (VB) and cultured in rotating bioreactors for up to 8 weeks. Structure (light and electron microscopy), biomechanical properties (interfacial adhesive strength, construct compressive modulus), biochemical composition (construct glycosaminoglycans (GAG), collagen, and cells), and adjacent tissue diffusivity were assessed. RESULTS: Engineered constructs were comprised predominately of hyaline cartilage, and appeared either closely apposed to adjacent cartilage or functionally interdigitated with adjacent bone due to interfacial deposition of extracellular matrix. An increase in culture time significantly improved construct adhesive strength (P<0.001), modulus (P=0.02), GAG (P=0.04) and cellularity (P<0.001). The type of adjacent tissue significantly affected construct adhesion (P<0.001), modulus (P<0.001), GAG (P<0.001) and collagen (P<0.001). For constructs cultured in rings of cartilage, negative correlations were observed between ring GAG content (log transformed) and construct adhesion (R2=0.66, P<0.005), modulus (R2=0.49, P<0.05) and GAG (R2=0.44, P<0.05). Integrative repair was better for constructs cultured adjacent to bone than cartilage, in association with its solid architectural structure and high GAG content, and best for constructs cultured adjacent to DB, in association with its high diffusivity. CONCLUSIONS: Chondrogenesis and integrative repair in engineered cartilage improved with time and depended on adjacent tissue architecture, composition, and transport properties.     

Cartilage tissue engineering by expanded goat articular chondrocytes.

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.     

Comparison of tracheal and nasal chondrocytes for tissue engineering of the trachea

BACKGROUND: This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage-derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced. METHODS: Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter x 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea. RESULTS: The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content. CONCLUSIONS: This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.     

Generation and characterization of osteochondral grafts with human nasal chondrocytes

We investigated whether nasal chondrocytes (NC) can be used to generate composite constructs with properties necessary for the repair of osteochondral (OC) lesions, namely maturation, integration and capacity to recover from inflammatory burst. OC grafts were fabricated by combining engineered cartilage tissues (generated by culturing NC or articular chondrocytes – AC – onto Chondro‐Gide^® matrices) with devitalized spongiosa cylinders (Tutobone^®). OC tissues were then exposed to IL‐1β for three days and cultured for additional 2 weeks in the absence of IL‐1β. Cartilage maturation extent was assessed (immune) histologically, biochemically and by delayed gadolinium‐enhanced magnetic resonance imaging of cartilage (dGEMRIC) while cartilage/bone integration was assessed using a peel‐off mechanical test. The use of NC as compared to AC allowed for more efficient cartilage matrix accumulation and superior integration of the cartilage/bone layers. dGEMRIC and biochemical analyzes of the OC constructs showed a reduced glycosaminoglycan (GAG) contents upon IL‐1β administration. Cartilaginous matrix contents and integration forces returned to baseline up on withdrawal of IL‐1β. By having a cartilage layer well developed and strongly integrated to the subchondral layer, OC tissues generated with NC may successfully engraft in an inflammatory post‐surgery joint environment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1111–1119, 2015.     

Failure of xenoimplantation using porcine synovium‐derived stem cell‐based cartilage tissue constructs for the repair of rabbit osteochondral defects

The use of xenogeneic tissues offers many advantages with respect to availability, quality control, and timing of tissue harvest. Our previous study indicated that implantation of premature tissue constructs from allogeneic synovium‐derived stem cells (SDSCs) facilitated cartilage tissue regeneration. The present study investigated the feasibility of xenoimplantation of SDSC‐based premature tissue constructs for the repair of osteochondral defects. Porcine SDSCs were mixed with fibrin gel, seeded in polyglycolic acid (PGA) scaffolds, and cultured in a rotating bioreactor system supplemented for 1 month with growth factor cocktails. The engineered porcine premature tissues were implanted to repair surgically induced osteochondral defects in the medial femoral condyles of 12 rabbits. Three weeks after surgery, the xenoimplantation group exhibited a smooth, whitish surface while the untreated control remained empty. Surprisingly, 6 months after surgery, the xenoimplantation group displayed some tissue loss while the untreated control group was overgrown with fibrocartilage tissue. In the xenoimplantation group, chronic inflammation was observed in synovial tissue where porcine major histocompatibility complex (MHC) class II antigen positively stained in the engulfed foreign bodies. In addition, porcine source cells also migrated from the implantation site and may have been responsible for the observed loss of glycosaminoglycans (GAGs) underneath surrounding articular cartilage. The histological score was much worse in the xenoimplanted group than in the untreated control. Our study suggested that SDSC‐based xenogeneic tissue constructs might cause delayed immune rejection. Xenotransplantation may not be an appropriate approach to repair osteochondral defects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1064–1070, 2010     

Sizable Scaffold‐Free Tissue‐Engineered Articular Cartilage Construct for Cartilage Defect Repair

This study introduces an implantable scaffold‐free cartilage tissue construct (SF) that is composed of chondrocytes and their self‐produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full‐thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16‐week old) and 3 week‐cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold‐free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold‐free engineered cartilage. When implanting tissue‐engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.     

新西兰大白兔骨膜下组织工程骨异位成骨的实验研究

目的：利用新西兰大白兔骨髓基质干细胞构建组织工程骨,研究其在股骨骨膜下异位成骨的可行性。方法：选取3月龄的清洁级雌性新西兰大白兔,体重3kg,取其骨髓基质干细胞诱导为成骨干细胞,扩增后接种到β-磷酸三钙生物陶瓷颗粒中,构建的组织工程骨种植到股骨骨膜下,3个月后实验动物血管内灌注凝胶墨汁,通过光学显微镜直接检测组织工程骨的血供和成骨结果。结果：16个标本中的12个植入的组织工程骨颗粒均良好固定在骨膜下并被骨膜包围,组织工程骨中有大量血管和新生骨形成,骨组织结构相对紊乱,不同于正常骨组织结构排列规则,血管分布均匀。4例取材时发现植入物游离于骨膜外,大部分材料被吸收,残留植入物体积明显小于骨膜下成骨,未见明显的骨组织形成,血供情况欠佳。股骨骨膜下组织工程骨颗粒80%贴附牢固,成骨良好,新骨内有大量血管长入。结论：组织工程骨可以在骨膜下获得良好的异位成骨。     

聚羟基丁酸酯载体人工软骨体内培育的实验研究

目的 探讨聚羟基丁酸酶（ＰＨＢ）泡沫材料作为软骨细胞支架材料以及体内培育组织工程化人工软骨的可行性。方法 取４周龄雄性新西兰幼免关节软骨、胶原酶消化,将所获软骨细胞种植到ＰＨＢ泡沫材料上,体外培养１周后,将细胞－支架材料复合体移植到兔背背部下皮,以单纯植入聚羟基丁酸酯泡沫材料及接种软骨细胞组为对照组。分别于术后第４、８、１２周取材,进行大体观察及组织学、免疫组织化学观察。结果 软骨细胞＝支架材料复     

体内外环境对组织工程软骨组织结构与力学性能的影响

目的 探讨体外构建组织工程化软骨在体、内外环境中力学性能及组织结构的变化,以及微环境对组织形成的影响,为工程化软骨构建提供适当参数。方法 体外培养扩增人胎儿关节软骨细胞,取第2代细胞以6×107个细胞/ml密度接种到聚羟基乙酸/聚乳酸(Polyglycolic acid/Polylactic acid,PGA/PLA)材料制成的圆柱形三维支架上,常规体外培养4周后,分为体内组(C、D组)和体外组（A、B组）,C、D组植于裸鼠皮下,A、B组继续常规培养液培养,于6、12周后取材,以正常软骨作为对照,行大体观察,以及组织学、组织化学、生物力学、超微结构等检测。结果 A、B、C、D4组均形成大体形态良好的透明软骨样组织。C、D组软骨呈乳白色,表面光滑,超微结构上胶原纤维排列致密而有规则,可形成有横纹的粗大胶原纤维,类似正常成人软骨;A、B组颜色偏黄,表面略粗糙,外观及超微结构近似半透明的胎儿关节软骨。工程化软骨植入体内12周后压缩弹性模量及胶原直径分别为（38.28±3.95）MPa和（41.58 ±2.78）nm,明显优于体外同时期组的(4.12±0.63) MPa和（15.83±1.70）nm(P ＜0.01)。结论 组织工程软骨的结构和功能在体内环境逐步成熟,体内组软骨超微结构上能形成粗大胶原纤维网络,胶原的交联增强可能是其力学性能较体外组明显提高的重要原因。     

Real-time Monitoring of Force Response Measured in Mechanically Stimulated Tissue-Engineered Cartilage

Abstract:  Mechanical stimulation improves tissue-engineered cartilage development both in terms of biochemical composition and structural properties. However, the link between the compositional changes attributed to mechanical stimulation and the changing structural properties of the engineered cartilage is poorly understood. We hypothesize that transient events associated with construct stiffening can be documented and used to understand milestones in construct development. To do this, we designed and built a mechanical stimulation bioreactor that can continuously record the force response of the engineered construct in real time. This study documents the transient changes of the stiffness of tissue-engineered cartilage constructs over the first 14 days of their development under cyclic loading. Compressive strain stimulation (15%, 1 Hz) was applied to poly(ethylene glycol) (PEG) hydrogels seeded with primary articular chondrocytes. The average compressive modulus of strain-stimulated constructs was 12.7 ± 1.45 kPa after 2 weeks, significantly greater ( P  < 0.01) than the average compressive moduli of both unstimulated constructs (10.7 ± 0.94 kPa) and nonviable stimulated constructs (11.2 ± 0.91 kPa). The system was able to document that nearly all of the stiffness increase occurred over the last 2 days of the experiment, where live-cell constructs demonstrated a rapid 20% increase in force response. The system's ability to track significant increases in stiffness over time was also confirmed by Instron testing. These results present a novel view of the early mechanical development of tissue-engineering cartilage constructs and suggest that the real-time monitoring of force response may be used to noninvasively track the development of engineered tissue.     

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction

Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). In this study, we hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of MSCs. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage‐like structure with positive Alcian blue staining in the cartilage fragment–fibrin–MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In conclusion, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:393–400, 2012