Mechanical stretch-induced changes in cell morphology and mRNA expression of tendon/ligament-associated genes in rat bone-marrow mesenchymal stem cells

It has been demonstrated that mechanical stimulation plays a vital role in regulating the proliferation and differentiation of stem cells. However, little is known about the effects of mechanical stress on tendon/ligament development from mesenchymal stem cells (MSCs). Here, using a custom-made cell-stretching device, we studied the effects of mechanical stretching on the cell morphology and mRNA expression of several key genes modulating tendon/ligament genesis. We demonstrate that bone-marrow-derived rat MSCs (rMSCs), when subjected to cyclic uniaxial stretching, express obvious detectable mRNAs for tenascin C and scleraxis, a unique maker of tendon/ligament formation, and significantly increased levels of type I collagen and type III collagen mRNAs. The stretched cells also orient at approximately 65 degrees with respect to the stretching direction and exhibit a more fibroblast-like morphology. Collectively, these results indicate that mechanical stretching facilitates the directed differentiation of rMSCs into tendon/ligament fibroblasts, which has potential implications for the tissue engineering of bioartificial tendons and ligaments.     

The effects of mechanical stretch on the biological characteristics of human adipose‐derived stem cells

Adipose‐derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs), which have promised a vast therapeutic potential in tissue regeneration. Recent studies have demonstrated that combining stem cells with mechanical stretch may strengthen the efficacy of regenerative therapies. However, the exact influences of mechanical stretch on MSCs still remain inconclusive. In this study, human ADSCs (hADSCs) were applied cyclic stretch stimulation under an in vitro stretching model for designated duration. We found that mechanical stretch significantly promoted the proliferation, adhesion and migration of hADSCs, suppressing cellular apoptosis and increasing the production of pro‐healing cytokines. For differentiation of hADSCs, mechanical stretch inhibited adipogenesis, but enhanced osteogenesis. Long‐term stretch could promote ageing of hADSCs, but did not alter the cell size and typical immunophenotypic characteristics. Furthermore, we revealed that PI3K/AKT and MAPK pathways might participate in the effects of mechanical stretch on the biological characteristics of hADSCs. Taken together, mechanical stretch is an effective strategy for enhancing stem cell behaviour and regulating stem cell fate. The synergy between hADSCs and mechanical stretch would most likely facilitate tissue regeneration and promote the development of stem cell therapy.     

Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors

Background  

Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings.     

Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, including vascular smooth muscle cells (SMCs), and have tremendous potential as a cell source for cardiovascular regeneration. We postulate that specific vascular environmental factors will promote MSC differentiation into SMCs. However, the effects of the vascular mechanical environment on MSCs have not been characterized. Here we show that mechanical strain regulated the expression of SMC markers in MSCs. Cyclic equiaxial strain downregulated SM alpha-actin and SM-22alpha in MSCs on collagen- or elastin-coated membranes after 1 day, and decreased alpha-actin in stress fibers. In contrast, cyclic uniaxial strain transiently increased the expression of SM alpha-actin and SM-22alpha after 1 day, which subsequently returned to basal levels after the cells aligned in the direction perpendicular to the strain direction. In addition, uniaxial but not equiaxial strain induced a transient increase of collagen I expression. DNA microarray experiments showed that uniaxial strain increased SMC markers and regulated the expression of matrix molecules without significantly changing the expression of the differentiation markers (e.g., alkaline phosphatase and collagen II) of other cell types. Our results suggest that uniaxial strain, which better mimics the type of mechanical strain experienced by SMCs, may promote MSC differentiation into SMCs if cell orientation can be controlled. This study demonstrates the differential effects of equiaxial and uniaxial strain, advances our understanding of the mechanical regulation of stem cells, and provides a rational basis for engineering MSCs for vascular tissue engineering and regeneration.     

Mechanical stimulation of tissue engineered tendon constructs: effect of scaffold materials

Our group has shown that numerous factors can influence how tissue engineered tendon constructs respond to in vitro mechanical stimulation. Although one study showed that stimulating mesenchymal stem cell (MSC)-collagen sponge constructs significantly increased construct linear stiffness and repair biomechanics, a second study showed no such effect when a collagen gel replaced the sponge. While these results suggest that scaffold material impacts the response of MSCs to mechanical stimulation, a well-designed intra-animal study was needed to directly compare the effects of type-I collagen gel versus type-I collagen sponge in regulating MSC response to a mechanical stimulus. Eight constructs from each cell line (n=8 cell lines) were created in specially designed silicone dishes. Four constructs were created by seeding MSCs on a type-I bovine collagen sponge, and the other four were formed by seeding MSCs in a purified bovine collagen gel. In each dish, two cell-sponge and two cell-gel constructs from each line were then mechanically stimulated once every 5 min to a peak strain of 2.4%, for 8 h/day for 2 weeks. The other dish remained in an incubator without stimulation for 2 weeks. After 14 days, all constructs were failed to determine mechanical properties. Mechanical stimulation significantly improved the linear stiffness (0.048+/-0.009 versus 0.015+/-0.004; mean+/-SEM (standard error of the mean ) N/mm) and linear modulus (0.016+/-0.004 versus 0.005+/-0.001; mean+/-SEM MPa) of cell-sponge constructs. However, the same stimulus produced no such improvement in cell-gel construct properties. These results confirm that collagen sponge rather than collagen gel facilitates how cells respond to a mechanical stimulus and may be the scaffold of choice in mechanical stimulation studies to produce functional tissue engineered structures.     

Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study

Mesenchymal stromal cells (MSCs) are considered to be an excellent source in regenerative medicine. They contain several cell subtypes, including multipotent stem cells. MSCs are of particular interest as they are currently being tested using cell and gene therapies for a number of human diseases. They represent a rare population in tissues; for this reason, they require, before being transplanted, an in vitro amplification. This process may induce replicative senescence, thus affecting differentiation and proliferative capacities. Increasing evidence suggests that MSCs from fetal tissues are significantly more plastic and grow faster than MSCs from bone marrow. Here, we compare amniotic fluid mesenchymal stromal cells (AF‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs) in terms of cell proliferation, surface markers, multidifferentiation potential, senescence, and DNA repair capacity. Our study shows that AF‐MSCs are less prone to senescence with respect to BM‐MSCs. Moreover, both cell models activate the same repair system after DNA damage, but AF‐MSCs are able to return to the basal condition more efficiently with respect to BM‐MSCs. Indeed, AF‐MSCs are better able to cope with genotoxic stress that may occur either during in vitro cultivation or following transplantation in patients. Our findings suggest that AF‐MSCs may represent a valid alternative to BM‐MSCs in regenerative medicine, and, of great relevance, the investigation of the mechanisms involved in DNA repair capacity of both AF‐MSCs and BM‐MSCs may pave the way to their rational use in the medical field.     

Comparing the Effect of Uniaxial Cyclic Mechanical Stimulation and Chemical Factors on Myogenin and Myh2 Expression in Mouse Embryonic and Bone Marrow Derived Mesenchymal Stem Cells

Background: Environmental factors affect stem cell differentiation. In addition to chemical factors, mechanical signals have been suggested to enhance myogenic differentiation of stem cells. Therefore, this study was undertaken to illustrate and compare the effect of chemical and mechanical stimuli on Myogenin (MyoG) and Myosin heavy chani 2 (Myh2) expression of mouse bone marrowderived mesenchymal stem cells (BMSCs) and embryonic stem cells (ESCs). Methods: After isolation and expansion of BMSCs and generation of embryoid bodies and spontaneous differentiation of ESCs, cells were examined in 4 groups: (1) control group: untreated cells; (2) chemical group: cells incubated in myogenic medium (5-azacythidine and horse serum for BMSCs, dimethyl sulfoxide (DMSO) and horse serum for ESCs) for 5 days; (3) mechanical group: cells exposed to uniaxial cyclic strain (8%, 1 Hz, 24 h) and (4) chemical + mechanical group: cells incubated in myogenic medium for 4 days and then exposed to uniaxial cyclic strain. Real-time PCR was used to examine the expression of MyoG and Myh2 as specific myogenic markers. Results: suggested that mechanical loading, as a single factor, could elevate MyoG and Myh2 expression. Combining chemical with mechanical factor increases expression and there was no significant difference in MyoG expression of ESCs- and MSCs-chemical + mechanical groups; however, Myh2 expression was significantly higher in ESCs-mechanical group than that in the same group of MSCs.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Mechano-regulation of mesenchymal stem cell differentiation and collagen organisation during skeletal tissue repair

A number of mechano-regulation theories have been proposed that relate the differentiation pathway of mesenchymal stem cells (MSCs) to their local biomechanical environment. During spontaneous repair processes in skeletal tissues, the organisation of the extracellular matrix is a key determinant of its mechanical fitness. In this paper, we extend the mechano-regulation theory proposed by Prendergast et al. (J Biomech 30(6):539–548, 1997) to include the role of the mechanical environment on the collagen architecture in regenerating soft tissues. A large strain anisotropic poroelastic material model is used in a simulation of tissue differentiation in a fracture subject to cyclic bending (Cullinane et al. in J Orthop Res 20(3):579–586, 2002). The model predicts non-union with cartilage and fibrous tissue formation in the defect. Predicted collagen fibre angles, as determined by the principal decomposition of strain- and stress-type tensors, are similar to the architecture seen in native articular cartilage and neoarthroses induced by bending of mid-femoral defects in rats. Both stress and strain-based remodelling stimuli successfully predicted the general patterns of collagen fibre organisation observed in vivo. This provides further evidence that collagen organisation during tissue differentiation is determined by the mechanical environment. It is envisioned that such predictive models can play a key role in optimising MSC-based skeletal repair therapies where recapitulation of the normal tissue architecture is critical to successful repair.     

Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells

The roles of various soluble factors in promoting the osteogenic differentiation of adult mesenchymal stem cells (MSCs) have been widely studied, but little is known about how the extracellular matrix (ECM) instructs the phenotypic transition between growth and differentiation. To investigate this question, we cultured MSCs on purified vitronectin or type-I collagen, motivated by our earlier tissue engineering work demonstrating that MSC adhesion to polymer scaffolds is primarily mediated by the passive adsorption of these two ECM ligands from serum. Using alkaline phosphatase activity and matrix mineralization as indicators of the early and late stages of osteogenesis, respectively, we report here that both substrates supported differentiation, but the mechanism was substrate dependent. Specifically, osteogenesis on vitronectin correlated with enhanced focal adhesion formation, the activation of focal adhesion kinase (FAK) and paxillin, and the diminished activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) pathways. By contrast, MSCs on type-I collagen exhibited reduced focal adhesion formation, reduced activation of FAK and paxillin, and increased activation of ERK and PI3K. Inhibition of ERK and FAK blocked mineral deposition on both substrates, suggesting that the observed differences in signaling pathways ultimately converge to the same cell fate. Understanding these mechanistic differences is essential to predictably control the osteogenic differentiation of MSCs and widen their use in regenerative medicine.     

Differential effects of cyclic uniaxial stretch on human mesenchymal stem cell into skeletal muscle cell

Both fetal and adult skeletal muscle cells are continually being subjected to biomechanical forces. Biomechanical stimulation during cell growth affects proliferation, differentiation and maturation of skeletal muscle cells. Bone marrow-derived hMSCs [human MSCs (mesenchymal stem cells)] can differentiate into a variety of cell types, including skeletal muscle cells that are potentially a source for muscle regeneration. Our investigations involved a 10% cyclic uniaxial strain at 1 Hz being applied to hMSCs grown on collagen-coated silicon membranes with or without IGF-I (insulin-like growth factor-I) for 24 h. Results obtained from morphological studies confirmed the rearrangement of cells after loading. Comparison of MyoD and MyoG mRNA levels between test groups showed that mechanical loading alone can initiate myogenic differentiation. Furthermore, comparison of Myf5, MyoD, MyoG and Myf6 mRNA levels between test groups showed that a combination of mechanical loading and growth factor results in the highest expression of myogenic genes. These results indicate that cyclic strain may be useful in myogenic differentiation of stem cells, and can accelerate the differentiation of hMSCs into MSCs in the presence of growth factor.     

Application of dedifferentiated fat cells for periodontal tissue regeneration

Periodontal diseases result from inflammation by bacterial infection in plaques, leading to tooth loss. However, regenerative approaches with periodontal tissue regeneration by guided tissue regeneration and enamel matrix derivative are not yet well established. Tissue regeneration requires three factors: cells, scaffold, and growth factors. Dedifferentiated fat cells (DFATs) are pluripotent with the same differentiation capacities as mesenchymal stem cells (MSCs). Access to MSCs is limited, whereas donor cells for DFATs are abundant in adipose tissues and can be non-invasively obtained. Therefore, we tested DFATs as a new source for periodontal tissue regeneration in an experimental periodontal tissue loss model in rats by transplanting DFATs on an atelocollagen scaffold using DFATs isolated from Sprague–Dawley (SD) rats expressing green fluorescent protein (GFP). GFP–DFAT cells were transplanted on the palatal side of the upper left first molar in SD rats and detected by H&E staining, GFP, and proliferating cell nuclear antigen (PCNA) expression. DFAT differentiation was also evaluated in three-dimensional cultures. GFP positive cells were detected in the regenerated tissue by the DFATs/scaffold mixture at 4 weeks after transplantation, and PCNA-positive cells were significantly increased in the periodontal ligament along the new bone in the DFATs/scaffold group more than in the scaffold-only group, suggesting that DFATs differentiate in the same manner as MSCs and regenerate in the defective areas. Consistent with previous reports, DFATs differentiation was slower than that with stem cells. The present study demonstrates that DFATs are pluripotent and an effective new source of cells for periodontal tissue regeneration.     

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo^{1, 2}.In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses³.Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques⁴.This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.Open in a separate windowClick here to view.^{(27M, flv)}     

In vitro and in vivo neurogenic potential of mesenchymal stem cells isolated from different sources

Regenerative medicine is an evolving interdisciplinary topic of research involving numerous technological methods that utilize stem cells to repair damaged tissues. Particularly, mesenchymal stem cells (MSCs) are a great tool in regenerative medicine because of their lack of tumorogenicity, immunogenicity and ability to perform immunomodulatory as well as anti-inflammatory functions. Numerous studies have investigated the role of MSCs in tissue repair and modulation of allogeneic immune responses. MSCs derived from different sources hold unique regenerative potential as they are self-renewing and can differentiate into chondrocytes, osteoblasts, adipocytes, cardiomyocytes, hepatocytes, endothelial and neuronal cells, among which neuronal-like cells have gained special interest. MSCs also have the ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation. In this review we focus on neural differentiation potential of MSCs isolated from different sources and how certain growth factors/small molecules can be used to derive neuronal phenotypes from MSCs. We also discuss the efficacy of MSCs when transplanted in vivo and how they can generate certain neurons and lead to relief or recovery of the diseased condition. Furthermore, we have tried to evaluate the appropriate merits of different sources of MSCs with respect to their propensity towards neurological differentiation as well as their effectiveness in preclinical studies.     

The effects of mechanical stress on the growth, differentiation, and paracrine factor production of cardiac stem cells

Stem cell therapies have been clinically employed to repair the injured heart, and cardiac stem cells are thought to be one of the most potent stem cell candidates. The beating heart is characterized by dynamic mechanical stresses, which may have a significant impact on stem cell therapy. The purpose of this study is to investigate how mechanical stress affects the growth and differentiation of cardiac stem cells and their release of paracrine factors. In this study, human cardiac stem cells were seeded in a silicon chamber and mechanical stress was then induced by cyclic stretch stimulation (60 cycles/min with 120% elongation). Cells grown in non-stretched silicon chambers were used as controls. Our result revealed that mechanical stretching significantly reduced the total number of surviving cells, decreased Ki-67-positive cells, and increased TUNEL-positive cells in the stretched group 24 hrs after stretching, as compared to the control group. Interestingly, mechanical stretching significantly increased the release of the inflammatory cytokines IL-6 and IL-1β as well as the angiogenic growth factors VEGF and bFGF from the cells in 12 hrs. Furthermore, mechanical stretching significantly reduced the percentage of c-kit-positive stem cells, but increased the expressions of cardiac troponin-I and smooth muscle actin in cells 3 days after stretching. Using a traditional stretching model, we demonstrated that mechanical stress suppressed the growth and proliferation of cardiac stem cells, enhanced their release of inflammatory cytokines and angiogenic factors, and improved their myogenic differentiation. The development of this in vitro approach may help elucidate the complex mechanisms of stem cell therapy for heart failure.     

Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

Introduction

Mesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).

Methods

Commercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.

Results

At 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.

Conclusion

This scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.     

Induction of chondrogenic differentiation in mesenchymal stem cells by TGF-beta cross-linked to collagen-PLLA [poly(l-lactic acid)] scaffold by transglutaminase 2

Transglutaminase-mediated cross-linking has been employed to optimize the mechanical properties and stability of tissue scaffolds. We have characterized tissue transglutaminase (TG2)-mediated cross-linking as a useful tool to deliver biologically-active TGF to mesenchymal stem cells (MSCs) and direct their differentiation towards a chondrogenic lineage. TGF-β3 is irreversibly cross-linked by TG2 to collagen type II-coated poly(l-lactic acid) nanofibrous scaffolds and activates Smad phosphorylation and Smad-dependent expression of a luciferase reporter. Human bone marrow-derived MSCs cultured on these scaffolds deposit cartilaginous matrix after 14 days of culture at 50 % efficiency compared to chondrogenesis in the presence of soluble TGF-β3. These findings are significant because they suggest a novel approach for the programming of MSCs in a spatially controlled manner by immobilizing biologically active TGF-β3 via cross-linking to a collagen-coated polymeric scaffold.     

Cytoskeletal changes and the system of regulation of alkaline phosphatase activity in human periodontal ligament cells induced by mechanical stress

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity.