Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold

Autologous mesenchymal stem cells (MSCs) have been used as a potential cell-based therapy in various animal and human diseases. Their differentiation capacity makes them useful as a novel strategy in the treatment of tissue injury in which the healing process is compromised or delayed. In horses, bone healing is slow, taking a minimum of 6–12 months. The osteogenic capacity of equine bone marrow and muscle MSCs mixed with fibrin glue or phosphate-buffered saline (PBS) as a scaffold is assessed. Bone production by the following groups was compared: Group 1, bone marrow (BM) MSCs in fibrin glue; Group 2, muscle (M) MSCs in fibrin glue; Group 3, BM MSCs in PBS; Group 4, M MSCs in PBS and as a control; Group 5, fibrin glue without cells. BM and M MSCs underwent osteogenic stimulation for 48 h prior to being injected intramuscularly into nude mice. After 4 weeks, the mice were killed and muscle samples were collected and evaluated for bone formation and mineralization by using radiology, histochemistry and immunohistochemistry. Positive bone formation and mineralization were confirmed in Group 1 in nude mice based on calcium deposition and the presence of osteocalcin and collagen type I; in addition, a radiopaque area was observed on radiographs. However, no evidence of mineralization or bone formation was observed in Groups 2–5. In this animal model, equine BM MSCs mixed with fibrin glue showed better osteogenic differentiation capacity compared with BM MSCs in PBS and M MSCs in either carrier.     

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.     

Osteogenic response to BMP-2 of hMSCs grown on apatite-coated scaffolds

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to non-mineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration.     

Different effects of intermittent and continuous fluid shear stresses on osteogenic differentiation of human mesenchymal stem cells

A reasonable mechanical microenvironment similar to the bone microenvironment in vivo is critical to the formation of engineering bone tissues. As fluid shear stress (FSS) produced by perfusion culture system can lead to the osteogenic differentiation of human mesenchymal stem cells (hMSCs), it is widely used in studies of bone tissue engineering. However, effects of FSS on the differentiation of hMSCs largely depend on the FSS application manner. It is interesting how different FSS application manners influence the differentiation of hMSCs. In this study, we examined the effects of intermittent FSS and continuous FSS on the osteogenic differentiation of hMSCs. The phosphorylation level of ERK1/2 and FAK is measured to investigate the effects of different FSS application manners on the activation of signaling molecules. The results showed that intermittent FSS could promote the osteogenic differentiation of hMSCs. The expression level of osteogenic genes and the alkaline phosphatase (ALP) activity in cells under intermittent FSS application were significantly higher than those in cells under continuous FSS application. Moreover, intermittent FSS up-regulated the activity of ERK1/2 and FAK. Our study demonstrated that intermittent FSS is more effective to induce the osteogenic differentiation of hMSCs than continuous FSS.     

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.     

Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.     

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.     

Fibrin gel improved the spatial uniformity and phenotype of human chondrocytes seeded on collagen scaffolds

A scaffold made of equine collagen type I based material has been assessed for its use in the preparation of tissue-engineered cartilage implants with human articular chondrocytes. Improvements of cell-seeding efficiency and specific gene expression were studied by combining solid scaffold with fibrin glue or human blood plasma. Following 3 weeks of static culture, mRNA expression levels of collagen type I, collagen type II, aggrecan and versican were analyzed by real-time quantitative PCR and compared to those in native cartilage and monolayer cell cultures.Constructs prepared with fibrin glue or plasma showed higher cell seeding efficiencies than those prepared without gel. Chondrocytes seeded directly onto a collagen scaffold appeared fibroblastic in shape while those encapsulated in fibrin gel were spherical. The presence of fibrin glue positively influences on mRNA levels of collagen type II and aggrecan, while blood plasma enhanced only the level of collagen type II expression. Levels of collagen type I and versican decreased in presence of fibrin glue.In orthopaedics, the combination of solid collagen fleece with fibrin gel for implant preparation is seen to be preferred over solid material or even cells in a suspension, since fibrin gel improves seeding capacity of the scaffold, supports equal distribution of cells and stimulates higher chondrogenic phenotype expression.     

The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.     

In vivo injectable human adipose tissue regeneration by adipose-derived stem cells isolated from the fluid portion of liposuction aspirates

Liposuction aspirates separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and isolated from the fluid portion termed liposuction aspirate fluid (LAF) cells, both of which contain adipose-derived stromal cells (ASCs). Here, we examined the biological differences between PLA and LAF cells and then tested the differentiation capacity of LAF cells in vivo. The cell surface marker and the multiple differentiation ability of fresh isolated PLA and LAF cells and which from passaged 3–5 were examined in vitro. LAF cells were then incubated in adipogenic medium, stained with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI), mixed with fibrin glue then injected to nude mice; fibrin glue without cells was as a control. Three months later, the transplants were subjected to macroscopic observation and histological analysis. PLA and LAF cells were similar in growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed increased expression of CD29, CD44, CD133 and HLA DR and decreased expression of CD34. In vivo differentiation assay showed the mixture of LAF cells and fibrin glue formed adipose tissue which contained red fluorescent DiI-positive adipocytes. LAF cells can be harvested more easily than PLA cells. The in vivo adipogenic capacity suggested LAF cells would be useful and valuable for cell-based therapies and soft tissue reconstruction.     

Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC’s from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.     

The in vivo assessment of a novel scaffold containing heparan sulfate for tissue engineering with human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and mature tissue macrophages, respectively. It was found that the survival of the implanted human cells was affected by the host response to the implant regardless of the presence of HS, highlighting the importance of controlling the host response to tissue engineering devices.     

Expression of proinflammatory cytokines by human mesenchymal stem cells in response to cyclic tensile strain

Mesenchymal stem cells produce proinflammatory cytokines during their normal growth. Direct or indirect regulation of bone resorption by these cytokines has been reported. However, the effects of osteogenic conditions—chemical and/or mechanical—utilized during in vitro bone tissue engineering on expression of cytokines by hMSCs have not been studied. In this study, we investigated the effects of cyclic tensile strain, culture medium (with and without dexamethasone), and culture duration on the expression of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and interleukin‐8 (IL‐8) by bone marrow derived human mesenchymal stem cells (hMSCs). Human MSCs seeded in three‐dimensional Type I collagen matrices were subjected to 0%, 10%, and 12% uniaxial cyclic tensile strains at 1 Hz for 4 h/day for 7 and 14 days in complete growth or dexamethasone‐containing osteogenic medium. Viability of hMSCs was maintained irrespective of strain level and media conditions. Expression of either TNF‐α or IL‐1β was not observed in hMSCs under any of the conditions investigated in this study. Expression of IL‐6 was dependent on culture medium. An increase in IL‐6 expression was caused by both 10% and 12% strain levels. Both 10% and 12% strain levels caused an increase in IL‐8 production by hMSCs that was dependent on the presence of dexamethasone. IL‐6 and IL‐8 expressions by hMSCs were induced by cyclic tensile strain and osteogenic differentiating media, indicating that IL‐6 and IL‐8 may be functioning as autocrine signals during osteogenic differentiation of hMSCs. J. Cell. Physiol. 219: 77–83, 2009. © 2008 Wiley‐Liss, Inc.     

A statistical model to allow the phasing out of the animal testing of demineralised bone matrix products

Demineralised bone matrix (DBM) products are complex mixtures of proteins known to influence bone growth, turnover, and repair. They are used extensively in orthopaedic surgery, and are bioassayed in vivo prior to being used in clinical applications. Many factors contribute to the osteogenic potency of DBM, but the relative contributions of these factors, as well as the possibility of interactive effects, are not completely defined. The "gold standard" measure of the therapeutic value of DBM, the in vivo assay for ectopic bone formation, is costly, time-consuming, and involves the use of numerous animal subjects. We have measured the levels of five growth factors released by the collagenase digestion of DBM, and statistically related these levels with osteogenic potency as determined by a standard in vivo model, in order to determine which value or combination of values of growth factors best predict osteogenic activity. We conclude that the level of BMP-2 is the best single predictor of osteogenic potency, and that adding the values of other growth factors only minimally increases the predictive power of the BMP-2 measurement. A small, but significant, interactive effect between BMP-2 and BMP-7 was demonstrated. We present a statistical model based on growth factor (e.g. BMP-2) analysis that best predicts the in vivo assay score for DBM. This model allows the investigator to predict which lots of DBM are likely to exhibit in vivo bioactivity and which are not, thus reducing the need to conduct in vivo testing of insufficiently active lots of DBM. This model uses cut-point analysis to allow the user to assign an estimate of acceptable uncertainty with respect to the "gold standard" test. This procedure will significantly reduce the number of animal subjects used to test DBM products.     

Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells

The purpose of this study was to determine whether a combination of fibrin glue and cultured periosteal cells will result in new bone formation at heterotopic sites in nude mice. Growing cells and developing matrices surrounding periosteal explants from the diaphyses of radii of newborn calves were minced and mixed with fibrin glue in a syringe. The cell/matrix-fibrin glue admixture was then injected into the subcutaneous space on the dorsum of athymic nude mice. After 12 weeks of implantation, gross morphology and histologic investigations showed newly formed bone structures in all cell/matrix-fibrin glue admixtures, but none in fibrin glue injected alone and used as control samples. Osteopontin, a protein important in bone development, was identified by a Western blot assay of the cell/matrix-fibrin glue composite. This study supports the feasibility of initiating site-directed formation of bone structures at heterotopic tissue sites by means of injection of cultured periosteal cells and matrix in a fibrin glue carrier.     

Perfusion regulation of hMSC microenvironment and osteogenic differentiation in 3D scaffold

The combination of hMSCs with 3D scaffolds has become an important approach to creating functional bone constructs. Bioreactors are important tools to mitigate mass transfer limitations and to provide controlled physiochemical and biomechanical environments for the 3D bone construct development. Media flow in the bioreactor systems is generally controlled either parallel or transverse with respect to the 3D construct, creating different cellular and biomechanical microenvironments in the 3D constructs. In this study, a custom designed modular perfusion bioreactor system was operated under either the parallel or transverse flow. The influence of the flow patterns on the characteristics of the hMSCs' cellular microenvironment and subsequent construct development was investigated. The parallel flow configuration retained ECM proteins and mitogenic growth factors within the scaffold, effectively preserving hMSC progenicity and proliferation potential (e.g., CFU-F, proliferation, and OCT-4), whereas the transverse flow induced hMSC osteogenic differentiation with higher ALP activity and calcium deposition and up-regulation of osteogenic bone markers (e.g., BMP-2, ALP, RUNX2, OSX, and OC). These results demonstrate the regulatory role of the macroscopic flow on the cellular microenvironment of the 3D hMSC construct, and suggest configuring media flow as a strategy for directing hMSC fate and 3D bone construct development in the perfusion bioreactor.