Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.     

Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes

Objectives: Chitosan is widely used as a scaffold for bone tissue engineering. However, up‐to‐date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan‐coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow. Materials and methods: Tissue culture plates coated with chitosan at different coating densities were used to evaluate the effects on hMSC adhesion and osteoblast differentiation. hMSCs were induced to differentiate into osteoblasts on the chitosan‐coated plates and were evaluated using established techniques: alkaline phosphatase assay, demonstration of presence of calcium and real time PCR. Results: The cells adhered to plates of lower coating density of chitosan, but formed viable cell aggregates at higher coating density (100 μg/sq.cm). Coating density of 25 μg/sq.cm, supporting cell adhesion was chosen for osteoblast differentiation experiments. Differentiating hMSCs showed higher mineral deposition and calcium content on chitosan‐coated plates. Chitosan upregulated genes associated with calcium binding and mineralization such as collagen type 1 alpha 1, integrin‐binding sialoprotein, osteopontin, osteonectin and osteocalcin, significantly. Conclusions: We demonstrate for the first time that chitosan enhanced mineralization by upregulating the associated genes. Thus, the study may help clinical situations promoting use of chitosan in bone mineralization, necessary for healing non‐union fractures and more.     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

冲击波诱导人骨髓基质细胞体内成骨研究

为了研究冲击波(SW)诱导人骨髓基质细胞(hMSCs)在动物体内成骨作用,根据前期工作结果,应用适宜能量冲击波(10kV,500次)处理体外培养的hMSCs,将SW组和对照组hMSCs与羟基磷灰石(HA)载体复合后体外培养2周,应用扫描电镜(SEM)检测细胞在载体表面的生长情况.将hMSCs-HA载体复合体植入裸鼠皮下,分别于术后4周、8周取材进行组织学、四环素荧光标记、SEM观察、碱性磷酸酶测定、RT-PCR检测骨钙素mRNA表达.结果表明,SW组及对照组细胞与HA载体体外复合后生长良好,且SW组细胞分泌较多的细胞基质;细胞载体复合体植入动物体内后,SW组载体表面有类骨组织形成,而对照组HA载体表面无骨组织形成;SW组与对照组的hMSCs-HA载体复合体碱性磷酸酶表达有显著性差异(P<0.01);SW组hMSCs-HA载体复合体术后4周与8周表达骨钙素mRNA,而对照组则无表达.提示hMSCs经适宜能量冲击波作用后与HA载体复合植入裸鼠体内具有成骨作用,适宜能量的冲击波作为一种新的促进hMSCs成骨分化的方法,可应用于组织工程领域.     

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.     

Biocompatibility evaluation of bioprinted decellularized collagen sheet implanted in vivo cornea using swept‐source optical coherence tomography

Corneal transplantation by full‐thickness penetrating keratoplasty with human donor tissue is a widely accepted treatment for damaged or diseased corneas. Although corneal transplantation has a high success rate, a shortage of high‐quality donor tissue is a considerable limitation. Therefore, bioengineered corneas could be an effective solution for this limitation, and a decellularized extracellular matrix comprises a promising scaffold for their fabrication. In this study, three‐dimensional bioprinted decellularized collagen sheets were implanted into the stromal layer of the cornea of five rabbits. We performed in vivo noninvasive monitoring of the rabbit corneas using swept‐source optical coherence tomography (OCT) after implanting the collagen sheets. Anterior segment OCT images and averaged amplitude‐scans were acquired biweekly to monitor corneal thickness after implantation for 1 month. The averaged cornea thickness in the control images was 430.3 ± 5.9 μm, while the averaged thickness after corneal implantation was 598.5 ± 11.8 μm and 564.5 ± 12.5 μm at 2 and 4 weeks, respectively. The corneal thickness reduction of 34 μm confirmed the biocompatibility through the image analysis of the depth‐intensity profile base. Moreover, hematoxylin and eosin staining supported the biocompatibility evaluation of the bioprinted decellularized collagen sheet implantation. Hence, the developed bioprinted decellularized collagen sheets could become an alternative solution to human corneal donor tissue, and the proposed image analysis procedure could be beneficial to confirm the success of the surgery.      

On‐demand three‐dimensional freeform fabrication of multi‐layered hydrogel scaffold with fluidic channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186. © 2009 Wiley Periodicals, Inc.     

The response of human mesenchymal stem cells to osteogenic signals and its impact on bone tissue engineering

Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.     

Promotion of human mesenchymal stem cell differentiation on bioresorbable polycaprolactone/biphasic calcium phosphate composite scaffolds for bone tissue engineering

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ～ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.     

Sustained Release of BMP-2 in Bioprinted Alginate for Osteogenicity in Mice and Rats

The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.     

Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair

The mechanical property of bone tissue scaffolds is one of the most important aspects in bone tissue engineering that has remained problematic. In our previous study, we fabricated a three‐dimensional scaffold from nano‐hydroxyapatite/gelatin (nHA/Gel) and investigated its efficiency in promoting bone regeneration both in vitro and in vivo. In the present study, the effect of adding silicon carbide (SiC) on the mechanical and biological behaviors of the nHA/Gel/SiC and bone regeneration in vivo were determined. nHA and SiC were synthesized and characterized by the X‐ray diffraction pattern and transmission electron microscope image. Layer solvent casting, freeze drying, and lamination techniques were applied to prepare these scaffolds. Then, the biocompatibility and cell adhesion behavior of the synthesized nHA/Gel/SiC scaffolds were investigated. For in vivo studies, rats were categorized into three groups: blank defect, blank scaffold, and rat bone marrow mesenchymal stem cells (rBM‐MSCs)/scaffold. After 1, 4, and 12 weeks post‐injury, the rats were sacrificed and the calvaria were harvested. Sections with a thickness of 5 µm thickness were prepared and stained with hematoxylin–eosin and Masson's Trichrome, and immunohistochemistry was performed. Our results showed that SiC effectively increased the mechanical properties of the nHA/Gel/SiC scaffold. No significant differences were observed in biocompatibility, cell adhesion, and cytotoxicity of the nHA/Gel/SiC in comparison with the nHA/Gel nanocomposite. Based on histological and immunohistochemical studies, both osteogenesis and collagenization were significantly higher in the rBM‐MSCs/scaffold group, quantitatively and qualitatively. The present study strongly suggests the potential of SiC as an alternative strategy to improve the mechanical and biological properties of bone tissue engineering scaffolds, and shows that the pre‐seeded nHA/Gel/SiC scaffold with rBM‐MSCs improves osteogenesis in the engineered bone implant.     

Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells

Osteoarthritis (OA) involves the degeneration of articular cartilage and subchondral bone. The capacity of articular cartilage to repair and regenerate is limited. A biodegradable, fibrous scaffold containing zinc oxide (ZnO) was fabricated and evaluated for osteochondral tissue engineering applications. ZnO has shown promise for a variety of biomedical applications but has had limited use in tissue engineering. Composite scaffolds consisted of ZnO nanoparticles embedded in slow degrading, polycaprolactone to allow for dissolution of zinc ions over time. Zinc has well-known insulin-mimetic properties and can be beneficial for cartilage and bone regeneration. Fibrous ZnO composite scaffolds, having varying concentrations of 1–10 wt.% ZnO, were fabricated using the electrospinning technique and evaluated for human mesenchymal stem cell (MSC) differentiation along chondrocyte and osteoblast lineages. Slow release of the zinc was observed for all ZnO composite scaffolds. MSC chondrogenic differentiation was promoted on low percentage ZnO composite scaffolds as indicated by the highest collagen type II production and expression of cartilage-specific genes, while osteogenic differentiation was promoted on high percentage ZnO composite scaffolds as indicated by the highest alkaline phosphatase activity, collagen production, and expression of bone-specific genes. This study demonstrates the feasibility of ZnO-containing composites as a potential scaffold for osteochondral tissue engineering.     

Fates and osteogenic differentiation potential of human mesenchymal stem cells in immunocompromised mice

Human mesenchymal stem cells (hMSCs) from bone marrow were genetically marked by using a murine leukaemia virus construct encoding enhanced green fluorescent protein (eGFP). The marked cells were either directly implanted into the tibialis anterior muscle or introduced into a variety of other tissue sites in immunocompromised mice (NOD/SCID and C.B-17 SCID/beige) to investigate their fates and differentiation potentials. It was observed that the hMSCs survived for up to 12 weeks and showed site-specific morphological phenotypes. hMSCs delivered by intravenous injection were found mainly in the lungs and were detected rarely in other organs. Histomorphometry showed that, after implantation of hMSCs into the tibialis anterior muscle juxtaskeletally, the areas of reactive host callus formation at 1 and 2 weeks and of ectopic human bone formation at 1 week were significantly increased compared with the control group. Expression of eGFP and human RUNX2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type I mRNAs were detected in mice implanted with the labelled hMSCs but not in sham-treated samples. Active clearance of the reactive callus and ectopic calcified tissue by osteoclast-like tartrate-resistant acid phosphatase-positive cells was observed. We conclude that the eGFP-labelled hMSCs can survive and retain the potential to differentiate morphologically into a variety of apparent mesenchymal phenotypes in vivo. Absolute confirmation of differentiation capacity requires further study and is complicated by known possibilities of fusion of donor and host cells or limited transfer of genetic material. Nevertheless, the genetically marked hMSCs are shown to participate extensively in bone formation and turnover. Control of the host osteoclast/macrophage responses resulting in clearance of formed osteogenic tissue warrants further investigation to promote prolonged human osteogenesis in immunocompromised mice. Furthermore, any proposed general cytotherapeutic strategy for enhanced osteogenesis is likely to require supplementation of local bone-forming biological signals.     

Donepezil hydrochloride as a novel inducer for osteogenic differentiation of mesenchymal stem cells on PLLA scaffolds in vitro

Over the past decades, bone defects caused by illness or trauma have been the most common traumatic injuries in humans and treatment of orthopedic infections has always been a serious challenge to experts in the world. In this project, poly L-lactic acid (PLLA) nanofibrous scaffolds were synthesized as a nontoxic, eco-friendly, and cost-effective scaffold by the electrospinning technique. Then, the impact of PLLA on the cell proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assayed in the presence and absence of donepezil hydrochloride (DH) which was prescribed in patients with Alzheimer's disease. Also, hMSCs were seeded on PLLA scaffold in the presence (PLLA-DH) and absence of 1 μg mL^-1 of DH under osteogenic induction media. Osteogenic differentiation of hMSCs was assessed by specific bone-related tests including alkaline phosphatase (ALP) activity, Alizarin red and von Kossa staining, calcium content assay. Also, Osteocalcin and osteopontin were evaluated as osteogenic proteins as well as ALP, osteonectin, osteocalcin, collagen type I (Col-I) and Runx2 as osteogenic genes via immunocytochemistry (ICC) and Real-time PCR analyses. The obtained data showed the higher ALP enzyme activity and biomineralization, more intensity during von Kossa staining as well as the increase in the expression rate of osteogenic related gene and protein markers in differentiated hMSCs on PLLA-DH. In conclusion, the present study revealed that the combination of PLLA scaffold with DH provides a scope to develop a suitable matrix in bone tissue engineering applications.     

Three-dimensional scaffold containing EGF incorporated biodegradable polymeric nanoparticles for stem cell based tissue engineering applications

Stem cell-based tissue engineering holds much hope for the development of multifunctional tissues to replace diseased organs. The attachment and survival of stem cells on a three-dimensional (3D) scaffold must be enhanced for faster progression of stem cell based tissue engineering. This study evaluate the stability of mesenchymal stem cells (MSCs) in 3D porous scaffolds composed of a collagen and chitosan blend impregnated with epidermal growth factor incorporated chitosan nanoparticles (EGF-CNP). The EGF-CNP scaffolds were characterized by transmission electron microscopy, which revealed that the nanoparticles were round in shape and 20 ∼ 50 nm in size. The scaffolds were prepared by freeze drying. A Fourier-transform infrared spectrum study revealed that the linkage between collagen and chitosan was through an ionic interaction. Thermal analysis and degradation studies showed that the scaffold could be used in tissue engineering application. MSCs proliferated well in the EGF-CNP impregnated scaffold. A scanning electron microscope study showed anchored and elongated MSCs on the EGF-CNP impregnated scaffold. A 3D biodegradable collagen chitosan scaffold impregnated with EGF-CNP is a promising transportable candidate for MSC-based tissue engineering, and this scaffold could be used as an in vitro model for subsequent clinical applications.     

Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have strong potential in regeneration of musculoskeletal tissues including cartilage and bone. The microenvironment, comprising of scaffold and soluble factors, plays a pivotal role in determining the efficacy of cartilage tissue regeneration from MSCs. In this study, we investigated the effect of a three-dimensional synthetic-biological composite hydrogel scaffold comprised of poly (ethylene glycol) (PEG) and chondroitin sulfate (CS) on chondrogenesis of MSCs. The cells in CS-based bioactive hydrogels aggregated in a fashion which mimicked the mesenchymal condensation and produced cartilaginous tissues with characteristic morphology and basophilic extracellular matrix production. The aggregation of cells resulted in an enhancement of both chondrogenic gene expressions and cartilage specific matrix production compared to control PEG hydrogels containing no CS-moieties. Moreover, a significant down-regulation of type X collagen expression was observed in PEG/CS hydrogels, indicating that CS inhibits the further differentiation of MSCs into hypertrophic chondrocytes. Overall, this study demonstrates the morphogenetic role of bioactive scaffold-mediated microenvironment on temporal pattern of cartilage specific gene expressions and subsequent matrix production during MSC chondrogenesis.     

Gene expression modulation in TGF‐β3‐mediated rabbit bone marrow stem cells using electrospun scaffolds of various stiffness

Tissue engineering has recently evolved into a promising approach for annulus fibrosus (AF) regeneration. However, selection of an ideal cell source, which can be readily differentiated into AF cells of various regions, remains challenging because of the heterogeneity of AF tissue. In this study, we set out to explore the feasibility of using transforming growth factor‐β3‐mediated bone marrow stem cells (tBMSCs) for AF tissue engineering. Since the differentiation of stem cells significantly relies on the stiffness of substrate, we fabricated nanofibrous scaffolds from a series of biodegradable poly(ether carbonate urethane)‐urea (PECUU) materials whose elastic modulus approximated that of native AF tissue. We cultured tBMSCs on PECUU scaffolds and compared their gene expression profile to AF‐derived stem cells (AFSCs), the newly identified AF tissue‐specific stem cells. As predicted, the expression of collagen‐I in both tBMSCs and AFSCs increased with scaffold stiffness, whereas the expression of collagen‐II and aggrecan genes showed an opposite trend. Interestingly, the expression of collagen‐I, collagen‐II and aggrecan genes in tBMSCs on PECUU scaffolds were consistently higher than those in AFSCs regardless of scaffold stiffness. In addition, the cell traction forces (CTFs) of both tBMSCs and AFSCs gradually decreased with scaffold stiffness, which is similar to the CTF change of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that tBMSCs had strong tendency to differentiate into various types of AF cells and presented gene expression profiles similar to AFSCs, thereby establishing a rationale for the use of tBMSCs in AF tissue engineering.