Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells

Recent evidences have suggested that humoral factors released from the appropriate co-cultured cells influenced the expansion and differentiation of mesenchymal stem cells (MSCs). However, little is known about the proliferation and differentiation of MSCs subjected to co-culture condition with tenocytes. In this study, we aimed to establish a co-culture system of MSCs and tenocytes and investigate the proliferation and tendon/ligament related gene expression of MSCs. MTT assay was used to detect the expansion of MSCs. Semi-quantitative RT-PCR was performed to investigate the expression of proliferation associated c-fos gene and tendon/ligament related genes, including type I collagen (Col I), type III collagen (Col III), tenascin C and scleraxis. Significant increase in MSCs expansion was observed after 3 days of co-culture with tenocytes. The c-fos gene expression was found distinctly higher than for control group on day 4 and day 7 of co-culture. The mRNA expression of four tendon/ligament related genes was significantly up-regulated after 14 days of co-culture with tenocytes. Thus, our research indicates that indirect co-culture with tenocytes promotes the proliferation and mRNA expression of tendon/ligament related genes in MSCs, which suggests a directed differentiation of MSCs into tendon/ligament.     

Indirect co-culture with tendons or tenocytes can program amniotic epithelial cells towards stepwise tenogenic differentiation

Background

Amniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue.

Methodology/Principal Findings

Freshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43).

Conclusions/Significance

The co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or explants. The co-cultural system can be proposed as a low cost and easy technique to engineer tendon for biological study and cell therapy approach.     

Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10⁻⁷ M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.     

Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells

Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration.     

Extracorporeal Shock Wave Treatment (ESWT) Improves In Vitro Functional Activities of Ruptured Human Tendon-Derived Tenocytes

In vitro models of human tenocytes derived from healthy as well as from ruptured tendons were established, characterized and used at very early passage (P1) to evaluate the effects of Extracorporeal Shock Wave Treatment (ESWT). The molecular analysis of traditional tenocytic markers, including Scleraxis (Scx), Tenomodulin (Tnm), Tenascin-C (Tn-C) and Type I and III Collagens (Col I and Col III), permitted us to detect in our samples the simultaneous expression of all these genes and allowed us to compare their levels of expression in relationship to the source of the cells and treatments. In untreated conditions, higher molecular levels of Scx and Col I in tenocytes from pathological compared to healthy samples have been detected, suggesting – in the cells from injured tendon – the natural trigger of an early differentiation and repairing program, which depends by Scx and requires an increase in collagen expression. When ESWT (at the dose of 0.14 mJ/mm²) was applied to cultured tenocytes explanted from injured source, Scx and Col I were significantly diminished compared to healthy counterpart, indicating that such natural trigger maybe delayed by the treatment, in order to promote cellular repair. Herein, we show for the first time that ESWT enhances in vitro functional activities of ruptured tendon-derived tenocytes, such as proliferation and migration, which could probably contributes to tendon healing in vivo.     

Effect of indomethacin and lactoferrin on human tenocyte proliferation and collagen formation in vitro

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in patients with injuries and inflammation of tendon and ligament, and as post-surgical analgesics. The aim of this study is to investigate the effect of indomethacin, a classic NSAID and its combinational effect with an anabolic agent of skeletal tissue, lactoferrin, on the proliferation and collagen formation of human tenocytes in vitro. A factorial experimental design was employed to study the dose-dependent effect of the combination of indomethacin and lactoferrin. The results showed that indomethacin at high concentration (100 μM) inhibited human tenocyte proliferation in culture medium with 1–10% fetal bovine serum (FBS) in vitro. Also, high dose of indomethacin inhibited the collagen formation of human tenocytes in 1% FBS culture medium. Lactoferrin at 50–100 μg/ml promoted human tenocyte survival in serum-free culture medium and enhanced proliferation and collagen synthesis of human tenocytes in 1% FBS culture medium. When 50–100 μg/ml lactoferrin was used in combination with 100–200 μM indomethacin, it partially rescued the inhibitory effects of indomethacin on human tenocyte proliferation, viability and collagen formation. To our knowledge, it is the first evidence that lactoferrin is anabolic to human tenocytes in vitro and reverses potential inhibitory effects of NSAIDs on human tenocytes.     

Flexor tendon wound healing in vitro: lactate up-regulation of TGF-beta expression and functional activity

Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming growth factor-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and 19 percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath fibroblasts demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.     

Fibroblast Growth Factor Inhibits Expression of Neural Markers in Cultures of Zebrafish Early Embryo Cells

Basic fibroblast growth factor (FGF) regulation of developmental markers in cell cultures derived from early zebrafish embryos was examined with the goal of in vitro culture of zebrafish embryonic stem cells and gaining an understanding of extracellular influences on early embryonic development. Markers were stem/primordial germ cell markers pou-2 and vas, neural markers zp-50, pax[zf-a], en-3, and wnt-1, and mesodermal markers gsc and myoD. Previously we had shown that FGF prevents the development of zebrafish pigment cells in vitro. In our culture system, FGF reduced expression of neural-specific markers, possibly implicating the FGF family in suppression of early neural cell development. Exposure to FGF for 24 hours at the time of seeding the cells was sufficient to suppress neural marker expression for a subsequent 4 days of culture, while absence of FGF for the first 24 hours of culture nullified the effect of FGF added subsequently. FGF predictably increased expression of gsc and myoD. Vas expression was unaffected, while pou-2 expression decreased with time in culture in the presence or absence of FGF. However, in situ hybridization identified a subpopulation of cells expressing pou-2, suggesting the possible continued existence of undifferentiated stem cells in the cultures. Received March 10, 2000; accepted May 10, 2000.     

CAPE promotes the expansion of human umbilical cord blood-derived hematopoietic stem and progenitor cells in vitro

Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.     

The effects of some natural products compared to synthetic products on the metabolic activity,proliferation, viability,migration, and wound healing in sheep tenocytes

BackgroundTendinopathy or tendon injuries can affect many people, causing a huge impact on their movements and maintaining standing posture. Treatment options include physiotherapy, anti-inflammatory drugs, and alternative medicine. The use of physiotherapy or anti-inflammatory drugs may cause some side effects like pain and liver failure, respectively, therefore, alternative medicine will be a better choice.MethodTenocytes were isolated from sheep Achilles tendon and used in Alamar blue assay to assess the metabolic activity, proliferation, and viability of tenocytes over 24 hrs. and 48 hrs., using natural and synthetic products [i.e., olive oil, oleic acid, corn oil, Inula viscosa oil, Inula viscosa extract, Nigella sativa oil, naproxen sodium, and paracetamol and LED photobiomodulation]. Furthermore, tenocytes viability was assessed by FDA/PI stain. For migration and healing of a wound, the scratch assay was used.ResultsAlamar blue assay over 24 hrs. showed that Nigella sativa oil increased the metabolic activity, proliferation, and viability of tenocytes significantly, while Alamar blue over 48 hrs. showed that oleic acid, LED, and their combination increased these parameters for tenocytes significantly. Olive oil increased the viability of tenocytes significantly using FDA/PI stains. Scratch assay revealed that Inula viscosa oil, Inula viscosa extract, and paracetamol increased tenocyte migration and healing significantly.ConclusionNigella sativa oil, olive oil, oleic acid, Inula viscosa oil, and Inula viscosa extract may be used as an alternative therapy for tendinopathy with less side effects.     

The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study

Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ∼10 fold) and tenomodulin (∼3–4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ∼7–11 fold; tenomodulin ∼4–5 fold) and non-tenocyte-related genes (∼3–8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ∼5–6 fold; tenomodulin ∼6–13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ∼28–50 fold; tenomodulin ∼14–48 fold) and non-tenocyte-related genes (2–5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.     

Embryonic mechanical and soluble cues regulate tendon progenitor cell gene expression as a function of developmental stage and anatomical origin

Stem cell-based engineering strategies for tendons have yet to yield a normal functional tissue, due in part to a need for tenogenic factors. Additionally, the ability to evaluate differentiation has been challenged by a lack of markers for differentiation. We propose to inform tendon regeneration with developmental cues involved in normal tissue formation and with phenotypic markers that are characteristic of differentiating tendon progenitor cells (TPCs). Mechanical forces, fibroblast growth factor (FGF)-4 and transforming growth factor (TGF)-β2 are implicated in embryonic tendon development, yet the isolated effects of these factors on differentiating TPCs are unknown. Additionally, developmental mechanisms vary between limb and axial tendons, suggesting the respective cell types are programmed to respond uniquely to exogenous factors. To characterize developmental cues and benchmarks for differentiation toward limb vs. axial phenotypes, we dynamically loaded and treated TPCs with growth factors and assessed gene expression profiles as a function of developmental stage and anatomical origin. Based on scleraxis expression, TGFβ2 was tenogenic for TPCs at all stages, while loading was for late-stage cells only, and FGF4 had no effect despite regulation of other genes. When factors were combined, TGFβ2 continued to be tenogenic, while FGF4 appeared anti-tenogenic. Various treatments elicited distinct responses by axial vs. limb TPCs of specific stages. These results identified tenogenic factors, suggest tendon engineering strategies should be customized for tissues by anatomical origin, and provide stage-specific gene expression profiles of limb and axial TPCs as benchmarks with which to monitor tenogenic differentiation of stem cells.     

Differential expression of Tenomodulin and Chondromodulin-1 at the insertion site of the tendon reflects a phenotypic transition of the resident cells

The insertion site of the tendon to the skeletal element is hypovascular and is one of the most common sites of dysfunction in the musculoskeletal system. However, the resident cells have been poorly defined due to a lack of a specific marker for tenocytes. We previously reported that Tenomodulin (Tnmd) and Chondromodulin-1 (Chm1) are homologous angiogenesis inhibitors and predominantly expressed in the avascular region of tendons and cartilage, respectively. In this study, we analyzed the expression of Tnmd, Chm1, alpha 1 chain of the type I collagen (Col1a1) and alpha 1 chain of the type II collagen (Col2a1) at the insertion site of the Achilles, patellar, or rotator cuff tendons of 1-week-old rabbits by in situ hybridization analysis. Tnmd was co-expressed with Col1a1 in tenocytes of these tendons, while Chm1 and Col2a1 were detected in chondrocytes of the hyaline cartilage. Interestingly, the cell population between Tnmd/Col1a1 positive tenocytes and Chm1/Col2a1 positive chondrocytes expressed Col1a1 but none of the other markers (Tnmd, Chm1, and Col2a1). Red blood cells were exclusively present at the interface between the tendon substance and cartilage in the insertion site of the Achilles tendon. Lack of Tnmd and Chm1 in this newly characterized cell population may allow the transitional zone between the poorly vascularized tendon and cartilage to establish the unique vascular pattern for blood supply.     

Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture

We have previously demonstrated that lineage negative cells (Lin^neg) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin^neg UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin^neg cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin^neg UCB cells, when appropriately cultured, are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation.     

Accumulation of Oxidized LDL in the Tendon Tissues of C57BL/6 or Apolipoprotein E Knock-Out Mice That Consume a High Fat Diet: Potential Impact on Tendon Health

ObjectiveClinical studies have suggested an association between dyslipidemia and tendon injuries or chronic tendon pain; the mechanisms underlying this association are not yet known. The objectives of this study were (1) to evaluate the impact of a high fat diet on the function of load-bearing tendons and on the distribution in tendons of oxidized low density lipoprotein (oxLDL), and (2) to examine the effect of oxLDL on tendon fibroblast proliferation and gene expression.MethodsGene expression (Mmp2, Tgfb1, Col1a1, Col3a1), fat content (Oil Red O staining), oxLDL levels (immunohistochemistry) and tendon biomechanical properties were examined in mice (C57Bl/6 or ApoE -/-) receiving a standard or a high fat diet. Human tendon fibroblast proliferation and gene expression (COL1A1, COL3A1, MMP2) were examined following oxLDL exposure.ResultsIn both types of mice (C57Bl/6 or ApoE -/-), consumption of a high fat diet led to a marked increase in oxLDL deposition in the load-bearing extracellular matrix of the tendon. The consumption of a high fat diet also reduced the failure stress and load of the patellar tendon in both mouse types, and increased Mmp2 expression. ApoE -/- mice exhibited more pronounced reductions in tendon function than wild-type mice, and decreased expression of Col1a1 compared to wild type mice. Human tendon fibroblasts responded to oxLDL by increasing their proliferation and their mRNA levels of MMP2, while decreasing their mRNA levels for COL1A1 and COL3A1.ConclusionThe consumption of a high fat diet resulted in deleterious changes in tendon function, and these changes may be explained in part by the effects of oxLDL, which induced a proliferative, matrix-degrading phenotype in human tenocytes.     

Gene expression profiles of cryopreserved CD34 human umbilical cord blood cells are related to their bone marrow reconstitution abilities in mouse xenografts

Human umbilical cord blood (UCB) cells are an alternative source of hematopoietic stem cells for treatment of leukemia and other diseases. It is very difficult to assess the quality of UCB cells in the clinical situation. Here, we sought to assess the quality of UCB cells by transplantation to immunodeficient mice. Cryopreserved CD34⁺ UCB cells from twelve different human donors were transplanted into sublethally irradiated NOD/shi-scid Jic mice. In parallel, the gene expression profiles of the UCB cells were determined from oligonucleotide microarrays. UCB cells from three donors failed to establish an engraftment in the host mice, while the other nine succeeded to various extents. Gene expression profiling indicated that 71 genes, including HOXB4, C/EBP-β, and ETS2, were specifically overexpressed and 23 genes were suppressed more than 2-fold in the successful UCB cells compared to those that failed. Functional annotation revealed that cell growth and cell cycle regulators were more abundant in the successful UCB cells. Our results suggest that hematopoietic ability may vary among cryopreserved UCB cells and that this ability can be distinguished by profiling expression of certain sets of genes.     

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold

BACKGROUND Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering.One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment,proliferation,and tenogenic differentiation of cells.However,there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in a collagen sponge-based 3D culture system.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We constructed a 3D culture system based on a type I collagen sponge scaffold.The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy.Primary BMSCs were isolated from Sprague-Dawley rats.Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay.The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot,respectively.The deposited collagen was assessed by Sirius Red staining.RESULTS Transforming growth factorβ1(TGF-β1)showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7(GDF-7)and insulin-like growth factor 1(IGF-1)in both the 2D and 3D cultures,and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment.In the 2D culture,the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1,IGF-1,or GDF-7 treatment.However,TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture.Strangely,we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1.Moreover,TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.     

Fibroblast Growth Factor Signaling Is Essential for Self-renewal of Dental Epithelial Stem Cells

A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.