Spontaneous Osteogenic Differentiation Research Articles

Spontaneous Osteogenic Differentiation of Human Mesenchymal Stem Cells by Tuna-Bone-Derived Hydroxyapatite Composites with Green Tea Polyphenol-Reduced Graphene Oxide.

In recent years, bone tissue engineering (BTE) has made significant progress in promoting the direct and functional connection between bone and graft, including osseointegration and osteoconduction, to facilitate the healing of damaged bone tissues. Herein, we introduce a new, environmentally friendly, and cost-effective method for synthesizing reduced graphene oxide (rGO) and hydroxyapatite (HAp). The method uses epigallocatechin-3-O-gallate (EGCG) as a reducing agent to synthesize rGO (E-rGO), and HAp powder is obtained from Atlantic bluefin tuna (Thunnus thynnus). The physicochemical analysis indicated that the E-rGO/HAp composites had exceptional properties for use as BTE scaffolds, as well as high purity. Moreover, we discovered that E-rGO/HAp composites facilitated not only the proliferation, but also early and late osteogenic differentiation of human mesenchymal stem cells (hMSCs). Our work suggests that E-rGO/HAp composites may play a significant role in promoting the spontaneous osteogenic differentiation of hMSCs, and we envision that E-rGO/HAp composites could serve as promising candidates for BTE scaffolds, stem-cell differentiation stimulators, and implantable device components because of their biocompatible and bioactive properties. Overall, we suggest a new approach for developing cost-effective and environmentally friendly E-rGO/HAp composite materials for BTE application.

FLIM imaging revealed spontaneous osteogenic differentiation of stem cells on gradient pore size tissue-engineered constructs

BackgroundThere is an urgent clinical need for targeted strategies aimed at the treatment of bone defects resulting from fractures, infections or tumors. 3D scaffolds represent an alternative to allogeneic MSC transplantation, due to their mimicry of the cell niche and the preservation of tissue structure. The actual structure of the scaffold itself can affect both effective cell adhesion and its osteoinductive properties. Currently, the effects of the structural heterogeneity of scaffolds on the behavior of cells and tissues at the site of damage have not been extensively studied.MethodsBoth homogeneous and heterogeneous scaffolds were generated from poly(L-lactic acid) methacrylated in supercritical carbon dioxide medium and were fabricated by two-photon polymerization. The homogeneous scaffolds consist of three layers of cylinders of the same diameter, whereas the heterogeneous (gradient pore sizes) scaffolds contain the middle layer of cylinders of increased diameter, imitating the native structure of spongy bone. To evaluate the osteoinductive properties of both types of scaffold, we performed in vitro and in vivo experiments. Multiphoton microscopy with fluorescence lifetime imaging microscopy was used for determining the metabolic states of MSCs, as a sensitive marker of cell differentiation. The results obtained from this approach were verified using standard markers of osteogenic differentiation and based on data from morphological analysis.ResultsThe heterogeneous scaffolds showed improved osteoinductive properties, accelerated the metabolic rearrangements associated with osteogenic differentiation, and enhanced the efficiency of bone tissue recovery, thereby providing for both the development of appropriate morphology and mineralization.ConclusionsThe authors suggest that the heterogeneous tissue constructs are a promising tool for the restoration of bone defects. And, furthermore, that our results demonstrate that the use of label-free bioimaging methods can be considered as an effective approach for intravital assessment of the efficiency of differentiation of MSCs on scaffolds.

A New Osteogenic Membrane to Enhance Bone Healing: At the Crossroads between the Periosteum, the Induced Membrane, and the Diamond Concept

The lack of viability of massive bone allografts for critical-size bone defect treatment remains a challenge in orthopedic surgery. The literature has reviewed the advantages of a multi-combined treatment with the synergy of an osteoconductive extracellular matrix (ECM), osteogenic stem cells, and growth factors (GFs). Questions are still open about the need for ECM components, the influence of the decellularization process on the latter, the related potential loss of function, and the necessity of using pre-differentiated cells. In order to fill in this gap, a bone allograft surrounded by an osteogenic membrane made of a decellularized collagen matrix from human fascia lata and seeded with periosteal mesenchymal stem cells (PMSCs) was analyzed in terms of de-/recellularization, osteogenic properties, PMSC self-differentiation, and angiogenic potential. While the decellularization processes altered the ECM content differently, the main GF content was decreased in soft tissues but relatively increased in hard bone tissues. The spontaneous osteogenic differentiation was necessarily obtained through contact with a mineralized bone matrix. Trying to deepen the knowledge on the complex matrix-cell interplay could further propel these tissue engineering concepts and lead us to provide the biological elements that allow bone integration in vivo.

Ternary MXene-loaded PLCL/collagen nanofibrous scaffolds that promote spontaneous osteogenic differentiation

Conventional bioinert bone grafts often have led to failure in osseointegration due to low bioactivity, thus much effort has been made up to date to find alternatives. Recently, MXene nanoparticles (NPs) have shown prominent results as a rising material by possessing an osteogenic potential to facilitate the bioactivity of bone grafts or scaffolds, which can be attributed to the unique repeating atomic structure of two carbon layers existing between three titanium layers. In this study, we produced MXene NPs-integrated the ternary nanofibrous matrices of poly(L-lactide-co-ε-caprolactone, PLCL) and collagen (Col) decorated with MXene NPs (i.e., PLCL/Col/MXene), as novel scaffolds for bone tissue engineering, via electrospinning to explore the potential benefits for the spontaneous osteogenic differentiation of MC3T3-E1 preosteoblasts. The cultured cells on the physicochemical properties of the nanofibrous PLCL/Col/MXene-based materials revealed favorable interactions with the supportive matrices, highly suitable for the growth and survival of preosteoblasts. Furthermore, the combinatorial ternary material system of the PLCL/Col/MXene nanofibers obviously promoted spontaneous osteodifferentiation with positive cellular responses by providing effective microenvironments for osteogenesis. Therefore, our results suggest that the unprecedented biofunctional advantages of the MXene-integrated PLCL/Col nanofibrous matrices can be expanded to a wide range of strategies for the development of effective scaffolds in bone tissue regeneration.Graphical

Long-term hypoxia inhibits the passage-dependent stemness decrease and senescence increase of human dental pulp stem cells

Dental pulp stem cells (DPSCs) derived from discarded orthodontic teeth are easily obtained and have become a promising source for mesenchymal stem cell-based therapy. However, the pulp tissue is limited, and long-term culture induces cell senescence. Hypoxic culture was expected to be suitable for DPSC expansion, but the results have been contradictory. The aim of this study was to verify the effect of hypoxic culture on human DPSCs (hDPSCs). hDPSCs were isolated and cultured in normoxic (ambient O2 concentration) and hypoxic (5% O2) environments from passage 3 (P3) to P6. The biological characteristics of the cells at P4 (short-term culture) and P6 (long-term culture) were evaluated, including the expression of surface markers, cellular proliferation activity, cellular senescence, and spontaneous and induced differentiation. The results showed that the morphology, phenotype, and proliferation activity of hDPSCs were not affected by hypoxic culture. Long-term normoxic culture of hDPSCs induced cell stemness loss and cell senescence, while hypoxic culture could alleviate these effects. The expression of the stemness markers STRO-1 and OCT4 was increased and the number of senescent cells and the expression of the senescence-related genes P53 and TGF-β were reduced by long-term hypoxic culture. Spontaneous osteogenic and adipogenic differentiation did not occur during long-term normoxic culture. However, hypoxic culture suppressed the expression of the osteogenic markers ALP and RUNX-2 and the adipogenic markers PPAR-γ and FABP4. The induced osteogenic and adipogenic differentiation was apparently reduced by hypoxic culture as well. Our findings indicate that long-term hypoxia culture is beneficial to the maintenance of hDPSCs’ biological characteristics and provide some insights into their large-scale expansion.

Melatonin Treatment Alters Biological and Immunomodulatory Properties of Human Dental Pulp Mesenchymal Stem Cells via Augmented Transforming Growth Factor Beta Secretion

IntroductionMelatonin is an endogenous neurohormone with well-reported anti-inflammatory and antioxidant properties, but the direct biological and immunomodulatory effects of melatonin on human dental pulp stem cells (hDPSCs) has not been fully elucidated. The aim of this study was to evaluate the influence of melatonin on the cytocompatibility, proliferation, cell migration, odontogenic differentiation, mineralized nodule formation, and immunomodulatory properties of hDPSCs. MethodsTo address the melatonin biological effects on hDPSCs, the cytocompatibility, proliferation, cell migration, odontogenic differentiation, mineralized nodule formation, and immunomodulatory properties of hDPSCs after melatonin treatment were evaluated. The statistical differences were evaluated using 1-way analysis of variance with the Tukey multiple comparison test. ResultsWe found that melatonin did not alter hDPSC immunophenotype or cell viability, even at the highest concentrations used. However, using intermediate melatonin concentrations (10–300 μmol/L), a significantly higher proliferation rate (P < .05 and P < .01) and migration of hDPSCs (P < .01) were observed. Importantly, melatonin treatment (100 μmol/L) significantly increased the secretion of the anti-inflammatory cytokine transforming growth factor beta (P < .05 and P < .01) and provoked a more robust antiproliferative effect on mitogen-stimulated T cells (P < .05). Finally, and unlike previous results found with mesenchymal stem cells from other sources, melatonin fails to induce or accelerate the spontaneous osteogenic differentiation of hDPSCs. ConclusionsTogether, these findings provide key data on the bioactivity of melatonin and its effects on hPDSC biological and immunomodulatory properties.

Osteogenic Potential of Decellularized Tooth Matrix.

We studied the possibility of using decellularized tooth matrix as a scaffold to restore bone tissue. It was found that mesenchymal stem cells underwent spontaneous osteogenic differentiation on the decellularized tooth matrix, which makes it possible to use it as a natural allograft in the treatment of resorption of alveolar bone tissue.

Murine Tissue-Resident PDGFRα+ Fibro-Adipogenic Progenitors Spontaneously Acquire Osteogenic Phenotype in an Altered Inflammatory Environment

Acquired heterotopic ossifications (HO) arising as a result of various traumas, including injury or surgical interventions, often result in pain and loss of motion. Though triggers for HO have been identified, the cellular source of these heterotopic lesions as well as the underlying mechanisms that drive the formation of acquired HO remain poorly understood, and treatment options, including preventative treatments, remain limited. Here, we explore the cellular source of HO and a possible underlying mechanism for their spontaneous osteogenic differentiation. We demonstrate that HO lesions arise from tissue-resident PDGFRα+ fibro/adipogenic progenitors (FAPs) in skeletal muscle and not from circulating bone marrow-derived progenitors. Further, we show that accumulation of these cells in the tissue after damage due to alterations in the inflammatory environment can result in activation of their inherent osteogenic potential. This work suggests a mechanism by which an altered inflammatory cell and FAP interactions can lead to the formation of HO after injury and presents potential targets for therapeutics in acquired HO. © 2020 American Society for Bone and Mineral Research.

Aminated 3D Printed Polystyrene Maintains Stem Cell Proliferation and Osteogenic Differentiation.

As 3D printing becomes more common and the technique is used to build culture platforms, it is imperative to develop surface treatments for specific responses. The advantages of aminating and oxidizing polystyrene (PS) for human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation are investigated. We find that ammonia (NH3) plasma incorporates amines while oxygen plasma adds carbonyl and carboxylate groups. Across 2D, 3D, and 3D dynamic culture, we find that the NH3- treated surfaces encouraged cell proliferation. Our results show that the NH3-treated scaffold was the only treatment allowing dynamic proliferation of hMSCs with little evidence of osteogenic differentiation. With osteogenic media, particularly in 3D culture, we find the NH3 treatment encouraged greater and earlier expression of RUNX2 and ALP. The NH3-treated PS scaffolds support hMSC proliferation without spontaneous osteogenic differentiation in static and dynamic culture. This work provides an opportunity for further investigations into shear profiling and coculture within the developed culture system toward developing a bone marrow niche model.

Injectable platelet-rich fibrin influences the behavior of gingival mesenchymal stem cells.

In this study, we examined the effects of injectable platelet-rich fibrin (iPRF) on proliferation and osteodifferentiation in mesenchymal stem cells (MSCs) isolated from human gingiva. Gingival MSCs (gMSCs) were grown in experimental culture media with different concentrations of iPRF [5%, 10%, and replacement of fetal calf serum (FCS) in the standard media with 10% iPRF–10% iPRF-FCS]. Immunophenotyping of gMSCs was performed after seven days by flow cytometry, and their proliferation was examined after three and seven days using the Cell Counting Kit-8 method. After 14 days in culture, spontaneous osteogenic differentiation of gMSCs was evaluated via real-time polymerase chain reaction. All gMSCs were positive for cluster of differentiation (CD) 105, CD73, CD90, and CD44, and negative for CD34/45, CD14, CD79a, and human leukocyte antigen, DR isotype (HLA-DR). Reduced expression of some surface antigens was observed in the gMSCs grown in 10% iPRF-FCS medium compared to the other groups. After three days, gMSCs grown in 10% iPRF had proliferated significantly less than the other groups. After seven days, proliferation was significantly higher in the 5% iPRF cells compared to the control, while proliferation in the 10% iPRF and 10% iPRF-FCS groups was significantly lower. No spontaneous osteogenic differentiation was observed in the presence of iPRF, as observed by low runt-related transcription factor 2 (RUNX2) expression. Some expression of secreted protein acidic and cysteine rich (SPARC) and collagen 1 alpha (COL1A) was observed for all the gMSCs regardless of the culture medium composition. gMSCs grown in 10% iPRF had significantly lower SPARC expression. In conclusion, 5% iPRF stimulated gMSC proliferation, and an excessively high concentration of iPRF can impair osteogenic induction.

Topographic cues of a novel bilayered scaffold modulate dental pulp stem cells differentiation by regulating YAP signalling through cytoskeleton adjustments.

ObjectivesTopographic cues can modulate morphology and differentiation of mesenchymal stem cells. This study aimed to determine how topographic cues of a novel bilayered poly (lactic‐co‐glycolic acid) (PLGA) scaffold affect osteogenic/odontogenic differentiation of dental pulp stem cells (DPSCs).MethodsThe surface morphology of the scaffolds was visualized by scanning electron microscope, and the surface roughness was measured by profilometry. DPSCs were cultured on each side of the scaffolds. Cell morphology, expression of Yes‐associated protein (YAP) and osteogenic/odontogenic differentiation were analysed by immunohistochemistry, real‐time polymerase chain reaction, and Alizarin Red S staining. In addition, cytochalasin D (CytoD), an F‐actin disruptor, was used to examine the effects of F‐actin on intracellular YAP localisation. Verteporfin, a YAP transcriptional inhibitor, was used to explore the effects of YAP signalling on osteogenic/odontogenic differentiation of DPSCs.ResultsThe closed side of our scaffold showed smaller pores and less roughness than the open side. On the closed side, DPSCs exhibited enhanced F‐actin stress fibre alignment, larger spreading area, more elongated appearance, predominant nuclear YAP localization and spontaneous osteogenic differentiation. Inhibition of F‐actin alignments was correlated with nuclear YAP exclusion of DPSCs. Verteporfin restricted YAP localisation to the cytoplasm, down‐regulated expression of early osteogenic/odontogenic markers and inhibited mineralization of DPSCs cultures.ConclusionsThe surface topographic cues changed F‐actin alignment and morphology of DPSCs, which in turn regulated YAP signalling to control osteogenic/odontogenic differentiation.

Hierarchically Porous Hydroxyapatite Hybrid Scaffold Incorporated with Reduced Graphene Oxide for Rapid Bone Ingrowth and Repair.

Hydroxyapatite (HA), the traditional bone tissue replacement material was widely used in the clinical treatment of bone defects because of its excellent biocompatibility. However, the processing difficulty and poor osteoinductive ability greatly limit the application of HA. Although many strategies have been reported to improve the machinability and osteointegration ability, the performance including mechanical strength, porosity, cell adhesion, etc. of material still can not meet the requirements. In this work, a soft template method was developed and a porous scaffold with hierarchical pore structure, nano surface morphology, suitable porosity and pore size, and good biomechanical strength was successfully prepared. The hierarchical pore structure is beneficial for cell adhesion, fluid transfer, and cell ingrowth. Moreover, the loaded reduced graphene oxide (rGO) can improve the adhesion and promote the proliferation and spontaneous osteogenic differentiation bone marrow mesenchymal stem cells. The scaffold is then crushed, degraded and wrapped by the newly formed bone and the newly formed bone gradually replaces the scaffold. The degradation rate of the scaffold well matches the rate of the new bone formation. The hierarchical porous HA/rGO composite scaffolds can greatly accelerate the bone ingrowth in the scaffold and bone repair in critical bone defects, thus providing a clinical potential candidate for large segment bone tissue engineering.

Argon plasma-treated fluorinated ethylene propylene: Growth of primary dermal fibroblasts and mesenchymal stem cells

Surface modification is an important step in making a synthetic polymer cytocompatible. We have previously reported improved cytocompatibility of immortalized human keratinocytes (HaCaT) with the otherwise bioinert fluorinated ethylene propylene (FEP) upon treatment with argon plasma discharge. In this article, we show that FEP modified with Ar plasma with the power of 3 and 8 W for 40 and 240 s served as a suitable material for cultivation of primary human dermal fibroblasts (HDF), which showed significantly improved proliferation and spreading comparable to standard tissue culture polystyrene. We also evaluated focal adhesions formed by HDF cells on modified FEP, which were far more numerous compared to pristine FEP. Moreover, we attempted spontaneous osteogenic differentiation of adipose-derived mesenchymal stem cells modified with human telomerase reverse transcriptase on Ar plasma-modified FEP. While the spontaneous osteogenic differentiation was unsuccessful, the cells were able to adhere and differentiated on tested matrices upon the administration of osteodifferentiation medium. These combined findings suggest that the treatment of FEP with Ar plasma comprises and efficient method to enable the adhesion and proliferation of various cell types on an otherwise largely bioinert material.

Poly(3-hydroxybutyrate)/poly(ethylene glycol) scaffolds with different microstructure: the effect on growth of mesenchymal stem cells.

Development of biocompatible 3D scaffolds is one of the most important challenges in tissue engineering. In this study, we developed polymer scaffolds of different design and microstructure to study cell growth in them. To obtain scaffolds of various microstructure, e.g., size of pores, we used double- and one-stage leaching methods using porogens with selected size of crystals. A composite of poly(3-hydroxybutyrate) (PHB) with poly(ethylene glycol) (PEG) (PHB/PEG) was used as polymer biomaterial for scaffolds. The morphology of scaffolds was analyzed by scanning electron microscopy; the Young modulus of scaffolds was measured by rheometry. The ability to support growth of mesenchymal stem cells (MSCs) in scaffolds was studied using the XTT assay; the phenotype of MSC was preliminarily confirmed by flow cytometry and the activity of alkaline phosphatase and expression level of CD45 marker was studied to test possible MSC osteogenic differentiation. The obtained scaffolds had different microstructure: the scaffolds with uniform pore size of about 125µm (normal pores) and 45µm (small pores) and scaffolds with broadly distributed pores size from about 50-100µm. It was shown that PHB/PEG scaffolds with uniform pores of normal size did not support MSCs growth probably due to their marked spontaneous osteogenic differentiation in these scaffolds, whereas PHB/PEG scaffolds with diverse pore size promoted stem cells growth that was not accompanied by pronounced differentiation. In scaffolds with small pores (about 45µm), the growth of MSC was the lowest and cell growth suppression was only partially related to stem cells differentiation. Thus, apparently, the broadly distributed pore size of PHB/PEG scaffolds promoted MSC growth in them, whereas uniform size of scaffold pores stimulated MSC osteogenic differentiation.

Graphene induces spontaneous osteogenic differentiation in vitro and in vivo

Graphene induces spontaneous osteogenic differentiation in vitro and in vivo

Oriented Surface Nanotopography Promotes the Osteogenesis of Mesenchymal Stem Cells

Stem cells could regulate their fate by responding to physical signals from their microenvironment. Recently, a few studies have shown that the spontaneous osteogenic differentiation of mesenchymal stem cells (MSCs) could occur on surfaces with nanoscaled structures (e.g., nanotubes and nanopits), which is essentially useful when designing biomaterial scaffolds for bone tissue engineering applications. However, the osteogenic nanotopography has not been widely investigated and the osteogenic mechanism of surfaces is still not well understood. In this study, oriented nanograting-like surfaces are fabricated by stretching the nanoholed inverse opal films, which are made through a convenient bottom-up approach of the self-assembly of colloidal nanoparticles. The effect of different surface nanotopography on the osteogenic differentiation of rat bone marrow-derived MSCs (rBMSCs) is examined in the absence of osteogenic inducing media and the underlying mechanisms are investigated using immunofluorescence staining and quantitative real-time polymerase chain reaction (PCR) analysis. It is found that rBMSCs on the oriented nanograting-like surfaces show significantly higher expression levels of osteogenic genes and more phosphorylation of Smad1/5, extracellular signal-regulated kinases (ERKs), and p38, confirming the activation of Smad and mitogen-activated protein kinase signaling pathways in the nanotopography-induced osteogenesis. Thus, the oriented nanograting-like surface nanotopography may provide a new strategy for the surface design of biomaterial scaffolds in bone tissue engineering.

Adipose-derived stem cells undergo spontaneous osteogenic differentiation in vitro when passaged serially or seeded at low density

Adipose-derived stem cells (ADSCs) are a convenient source of cells for regenerating tissue. Widespread application of ADSCs requires that they propagate efficiently and differentiate in vitro. We investigated the differentiation potential of ADSCs during long-term expansion in vitro and when the cells were seeded at low density. ADSCs were isolated from the inguinal fat pads of 3-week-old male rats, then cultured serially for 12 passages; some ADSCs at passage 3 were seeded at low density. The differentiation potential of ADSCs from passage 3 to passage 12 was assessed by their capacity for adipogenesis and osteogenesis while cultured in specific induction media. Spontaneous osteogenesis of ADSCs at passage 12 and of ADSCs that were seeded at low density was detected by western blotting, alizarin red S staining and measurement of alkaline phosphatase (ALP) activity. We found that with increasing passage number, the adipogenic potential of ADSCs decreased and osteogenic differentiation increased. Alizarin red S staining, bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (Runx2) expressions, and ALP activity demonstrated that both ADSCs at passage 12 and those that were seeded at low density differentiated into osteoblasts without additional induction factors.

Spontaneous osteogenic differentiation of mesenchymal stem cells on electrospun nanofibrous scaffolds

Hydroxyapatite-containing PHBV nanofibrous scaffolds accelerate osteogenic differentiation of MSCs by activating the related signaling pathways.

Reduced graphene oxide-coated hydroxyapatite composites stimulate spontaneous osteogenic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite the potential biomedical applications of graphene and its derivatives, only limited information is available regarding their osteogenic activity. This study concentrates upon the effects of reduced graphene oxide (rGO)-coated hydroxyapatite (HAp) composites on osteogenic differentiation of hMSCs. The average particle sizes of HAp and rGO were 1270 ± 476 nm and 438 ± 180 nm, respectively. When coated on HAp particulates, rGO synergistically enhanced spontaneous osteogenic differentiation of hMSCs, without hampering their proliferation. This result was confirmed by determining alkaline phosphatase activity and mineralization of calcium and phosphate as early and late stage markers of osteogenic differentiation. It is suggested that rGO-coated HAp composites can be effectively utilized as dental and orthopedic bone fillers since these graphene-based particulate materials have potent effects on stimulating the spontaneous differentiation of MSCs and show superior bioactivity and osteoinductive potential.

Transplantation of Fetal Instead of Adult Fibroblasts Reduces the Probability of Ectopic Ossification During Tendon Repair

Although cell transplantation therapy can effectively promote functional tendon repair, occasional ectopic ossification during tendon regeneration undermines its efficacy. The effect of transplanted cell types on ectopic ossification has not yet been systematically evaluated. This study compared the rate of ectopic ossification during tendon repair upon transplantation with mouse fetal fibroblasts (FFs) and their adult counterparts (adult fibroblasts [AFs]). Alkaline phosphatase (ALP) staining, immunofluorescence, and gene expression analysis were used to compare the spontaneous osteogenic differentiation of FFs and AFs in vitro. X-ray, histology, and gene expression analysis were used to investigate the ectopic ossification in a mouse Achilles tendon repair model in vivo. ALP staining and immunofluorescence data in vitro showed that FFs had less spontaneous osteogenic differentiation capacity, and lower expression of runt-related transcription factor 2 (runx2). For the in vivo study, the FFs transplant group displayed reduced ectopic ossification (2/7 vs. 7/7, Mann-Whitney test p<0.01) at 14 weeks post-transplantation and enhanced tendon repair (general histological score at week 6, 7.53 vs. 10.56, p<0.05). More chondrocytes formed at 6 weeks, and all mice developed bone marrow at 14 weeks post-transplantation in the AFs transplant group. Gene expression analysis of the regenerated tissue showed significantly higher expression levels of transforming growth factor beta1 (TGF-β1) and transforming growth factor beta3 (TGF-β3) in the AFs group during the early stages of tendon repair. Our study demonstrates that transplantation of fetal instead of AFs is more promising for tendon repair, underscoring the importance of the origin of seed cells for tendon repair.