Cultured cell‐derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells

Cell‐derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro‐environment. Therefore, cultured cell‐derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro‐environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell‐derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)‐derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.     

Osteogenic capacity of human BM‐MSCs,AT‐MSCs and their co‐cultures using HUVECs in FBS and PL supplemented media

Human bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and human adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) are the most frequently used stem cells in tissue engineering. Due to major clinical demands, it is necessary to find an optimally safe and efficient way for large‐scale expansion of these cells. Considering the nutritional source in the culture medium and method, this study aimed to analyze the effects of FBS‐ and PL‐supplemented media on osteogenesis in stem cell mono‐ and co‐cultures with human umbilical vein endothelial cells (HUVECs). Results showed that cell metabolic activity and proliferation increased in PL‐ compared to FBS‐supplemented media in mono‐ and co‐cultures for both BM‐MSCs and AT‐MSCs. In addition, calcium deposition was cell type dependent and decreased for BM‐MSCs but increased for AT‐MSCs in PL‐supplemented medium in both mono‐ and co‐cultures. Based on the effects of co‐cultures, BM‐MSCs/HUVECs enhanced osteogenesis compared to BM‐MSCs monocultures in both FBS‐ and PL‐supplemented media whereas AT‐MSCs/HUVECs showed similar results compared to AT‐MSCs monocultures. Copyright © 2013 John Wiley & Sons, Ltd.     

Three‐dimensional co‐culture of human hepatocytes and mesenchymal stem cells: improved functionality in long‐term bioreactor cultures

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short‐term viability in two‐dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co‐cultured with human bone marrow mesenchymal stem cells (BM‐MSCs) as spheroids in automated, computer‐controlled, stirred‐tank bioreactors with perfusion operation mode. A dual‐step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM‐MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co‐cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated‐dose toxicity testing. Moreover, the excretory activity was maintained in co‐cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM‐MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Engineering cartilaginous grafts using chondrocyte‐laden hydrogels supported by a superficial layer of stem cells

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co‐cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self‐assembled on top of CC‐laden agarose gels (structured co‐culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co‐culture). Porcine infrapatellar fat pad‐derived stem cells (FPSCs) and bone marrow‐derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co‐culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co‐culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co‐culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co‐culture for both MSC sources. In conclusion, assembling MSCs onto CC‐laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd.     

Agonism of Wnt–β‐catenin signalling promotes mesenchymal stem cell (MSC) expansion

Promoting mesenchymal stem cell (MSC) proliferation has numerous applications in stem cell therapies, particularly in the area of regenerative medicine. In order for cell‐based regenerative approaches to be realized, MSC proliferation must be achieved in a controlled manner without compromising stem cell differentiation capacities. Here we demonstrate that 6‐bromoindirubin‐3′‐oxime (BIO) increases MSC β‐catenin activity 106‐fold and stem cell‐associated gene expression ~33‐fold, respectively, over untreated controls. Subsequently, BIO treatment increases MSC populations 1.8‐fold in typical 2D culture conditions, as well as 1.3‐fold when encapsulated within hydrogels compared to untreated cells. Furthermore, we demonstrate that BIO treatment does not reduce MSC multipotency where MSCs maintain their ability to differentiate into osteoblasts, chondrocytes and adipocytes using standard conditions. Taken together, our results demonstrate BIO's potential utility as a proliferative agent for cell transplantation and tissue regeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

Cartilage graft engineering by co‐culturing primary human articular chondrocytes with human bone marrow stromal cells

Co‐culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small‐scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold‐based constructs generated by combining primary human ACs with human bone marrow MSCs (BM‐MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM‐MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co‐culture of ACs (25%) with BM‐MSCs (75%) in scaffolds resulted in up to 1.4‐fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8‐fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM‐MSCs with a relatively low fraction of non‐expanded ACs in porous scaffolds. The proposed co‐culture strategy is directly relevant towards a single‐stage surgical procedure for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of mesenchymal stem cells on stomach tissue engineering using small intestinal submucosa

Small intestinal submucosa (SIS) is a biodegradable collagen‐rich matrix containing functional growth factors. We have previously reported encouraging outcomes for regeneration of an artificial defect in the rodent stomach using SIS grafts, although the muscular layer was diminutive. In this study, we investigated the feasibility of SIS in conjunction with mesenchymal stem cells (MSCs) for regeneration of the gastrointestinal tract. MSCs from the bone marrow of green fluorescence protein (GFP)‐transgenic Sprague–Dawley (SD) rats were isolated and expanded ex vivo. A 1 cm whole‐layer stomach defect in SD rats was repaired using: a plain SIS graft without MSCs (group 1, control); a plain SIS graft followed by intravenous injection of MSCs (group 2); a SIS graft co‐cultured with MSCs (group 3); or a SIS sandwich containing an MSC sheet (group 4). Pharmacological, electrophysiological and immunohistochemical examination was performed to evaluate the regenerated stomach tissue. Contractility in response to a muscarinic receptor agonist, a nitric oxide precursor or electrical field stimulation was observed in all groups. SIS grafts seeded with MSCs (groups 3 and 4) appeared to support improved regeneration compared with SIS grafts not seeded with MSCs (groups 1 and 2), by enabling the development of well‐structured smooth muscle layers of significantly increased length. GFP expression was detected in the regenerated interstitial tissue, with fibroblast‐like cells in the seeded‐SIS groups. SIS potently induced pharmacological and electrophysiological regeneration of the digestive tract, and seeded MSCs provided an enriched environment that supported tissue regeneration by the SIS graft in the engineered stomach. © 2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

Glottic regeneration with a tissue‐engineering technique,using acellular extracellular matrix scaffold in a canine model

Acellular extracellular matrix scaffold derived from porcine urinary bladder (UBM) is decellularized material that has shown success for constructive remodelling of various tissues and organs. The regenerative effects of UBM were reported for the tympanic membrane, oesophagus, trachea, larynx, pleura and pericardium in animal studies, with promising results. The aim of this study was to investigate the regenerative effects of UBM on hemilarynx, using a canine model. A left partial hemilaryngectomy was performed and the surgical defects were reconstructed by insertion of UBM scaffold. Although local infection was observed in one dog in 1 week after implantation of the scaffold, all dogs showed good re‐epithelialization with minimum complication in 1 month. The effect of regeneration of the larynx was evaluated 6 months after the operation. The excised larynx experiments were performed to measure phonation threshold pressure (PTP), normalized mucosal wave amplitude (NMWA) and normalized glottal gap (NGG). The results of the measurements showed that PTP was normal or near normal in two cases and NMWA was within normal range in three cases, although there were individual variations. Histological examination was completed to evaluate structural changes in the scaffold with the appearance of the new cartilaginous structure. However, the regenerated vocal fold mucosa was mostly scarred. The UBM scaffold has shown to be biocompatible, biodegradable and useful for tissue regeneration of the hemilarynx, with possible restoration of function of the vocal fold. The vocal fold mucosa was scarred, which is the next challenge to be addressed. Copyright © 2014 John Wiley & Sons, Ltd.     

Biomimetic scaffolds and dynamic compression enhance the properties of chondrocyte‐ and MSC‐based tissue‐engineered cartilage

Adult chondrocytes are surrounded by a protein‐ and glycosaminoglycan‐rich extracellular matrix and are subjected to dynamic mechanical compression during daily activities. The extracellular matrix and mechanical stimuli play an important role in chondrocyte biosynthesis and homeostasis. In this study, we aimed to develop scaffold and compressive loading conditions that mimic the native cartilage micro‐environment and enable enhanced chondrogenesis for tissue engineering applications. Towards this aim, we fabricated porous scaffolds based on silk fibroin (SF) and SF with gelatin/chondroitin sulfate/hyaluronate (SF‐GCH), seeded the scaffolds with either human bone marrow mesenchymal stromal cells (BM‐MSCs) or chondrocytes, and evaluated their performance with and without dynamic compression. Human chondrocytes derived from osteoarthritic joints and BM‐MSCs were seeded in scaffolds, precultured for 1 week, and subjected to compression with 10% dynamic strain at 1 Hz, 1 hr/day for 2 weeks. When dynamic compression was applied, chondrocytes significantly increased expression of aggrecan (ACAN) and collagen X (COL10A1) up to fivefold higher than free‐swelling controls. In addition, dynamic compression dramatically improved the chondrogenesis and chondrocyte biosynthesis cultured in both SF and SF‐GCH scaffolds evidenced by glycosaminoglycan (GAG) content, GAG/DNA ratio, and immunostaining of collagen type II and aggrecan. However, both chondrocytes and BM‐MSCs cultured in SF‐GCH scaffolds under dynamic compression showed higher GAG content and compressive modulus than those in SF scaffolds. In conclusion, the micro‐environment provided by SF‐GCH scaffolds and dynamic compression enhances chondrocyte biosynthesis and matrix accumulation, indicating their potential for cartilage tissue engineering applications.     

Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow‐derived MSCs in a co‐culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co‐culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co‐cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down‐regulated in co‐cultures on polystyrene, they were up‐regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co‐culture, direct cell‐cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells. Copyright © 2012 John Wiley & Sons, Ltd.     

TGFβ3 secretion by three‐dimensional cultures of human dental apical papilla mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less‐studied MSC source. These dental‐derived MSCs are of great interest because of their potential as an accessible source for cell‐based therapies and tissue‐engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic‐mediated repair and regenerative effects observed when they are implanted. TGFβ3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP‐derived MSCs (hSCAPs) can produce and secrete TGFβ3 in response to micro‐environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFβ3 secretion. We found that dynamic changes of cell–matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two‐dimensional, 2D) towards a three‐dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFβ3 secretion, while monolayer cultures showed almost 10‐fold less secretion of TGFβ3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

The efficacy of a novel collagen–gelatin scaffold with basic fibroblast growth factor for the treatment of vocal fold scar

Vocal fold scar remains a therapeutic challenge. Basic fibroblast growth factor (bFGF) was reported to have regenerative effects for vocal fold scar, although it has the disadvantage of rapid absorption in vivo. A collagen–gelatin sponge (CGS) can compensate for the disadvantage by providing a sustained release system. The current study evaluated the efficacy of CGS combined with bFGF on vocal fold scar, using rat fibroblasts for an in vitro model and a canine in vivo model. We prepared fibroblasts from scarred vocal folds (sVFs) in rats and showed that bFGF accelerated cell proliferation and suppressed expression levels of cleaved caspase 3 and α‐smooth muscle actin. Has 1, Has 3, Fgf2, Hgf and Vegfa mRNA levels were significantly upregulated, while Col1a1 and Col3a1 were dose‐dependently downregulated, with a maximum effect at 100 ng/ml bFGF. In an in vivo assay, 6 weeks after lamina propria stripping, beagles were divided into three groups: CGS alone (CGS group); CGS with bFGF (7 µg/cm²; CGS + bFGF group); or a sham‐treated group. Vibratory examination revealed that the glottal gap was significantly reduced in the bFGF group and the two implanted groups, whereas the CGS + bFGF group showed higher mucosal wave amplitude. Histological examination revealed significantly restored hyaluronic acid and elastin redistribution in the CGS + bFGF group and reductions in dense collagen deposition. These results provide evidence that CGS and bFGF combination therapy may have therapeutic potential and could be a promising tool for treating vocal fold scar. Copyright © 2015 John Wiley & Sons, Ltd.     

Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear‐cut effects on cell proliferation,differentiation and metabolism

Physiologically low O₂ tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0–5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O₂ tensions (3–10%). We hypothesized that O₂ concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34⁺‐enriched cells was studied in co‐culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O₂. 2% O₂ resulted in a significantly lower CD34⁺ cell expansion (25‐fold vs 60‐, 64‐ and 92‐fold at day 10 for 5%, 21%, 10% O₂, respectively). In turn, 10% O₂ promoted the highest CD34⁺CD90⁺ cell expansion, reaching 22 ± 5.4‐ vs 5.6 ± 2.4‐ and 5.7 ± 2.0‐fold for 2%, 5% and 21% O₂, respectively, after 14 days. Similar differentiation patterns were observed under different O₂ tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O₂. Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O₂ when compared to higher O₂ concentrations (5–21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O₂, can maximize the ex vivo expansion of UCB stem/progenitor cells in co‐culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid method for obtaining mesenchymal stem cells and platelets from bone marrow aspirate

Mesenchymal stem cells (MSCs) and platelet‐rich plasma (PRP) are currently used alone or in combination for therapeutic applications especially for bone repair. We tested whether MSCs can be isolated from bone marrow (BM) aspirate using a commercially available kit commonly used to obtain PRP from peripheral blood (PB). Results revealed that mononuclear cells and platelets from both PB and BM could be efficiently isolated by obtaining a mononuclear and platelet rich fraction (PB‐MPRF and BM‐MPRF, respectively). Starting with comparable volumes, the number of platelets increased 1.5‐fold in BM‐MPRF compared to PB‐MPRF. The number of clonogenic cells in BM‐MPRF samples was significantly higher than whole BM samples as revealed by CFU‐F assay (54.92 ± 8.55 CFU‐F/1.5 x 10⁵ nucleated cells and 32.50 ± 12.43 CFU‐F/1.5 x 10⁵ nucleated cells, respectively). Cells isolated from BM‐MPRF after in vitro expansion fulfilled the definition of MSCs by phenotypic criteria, and differentiated along osteogenic, adipogenic and chondrogenic lineages following induction. Results showed that the kit isolated MSCs and platelets from BM aspirate. Isolated MSCs were further expanded in a laboratory and BM‐MPRF was used clinically following BM withdrawal for rapid intra‐operative cell therapy for the treatment of bone defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Matrix formation is enhanced in co‐cultures of human meniscus cells with bone marrow stromal cells

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co‐cultured in vitro in three‐dimensional (3D) pellet culture at three different cell–cell ratios for 3 weeks under normal (21% O₂) or low (3% O₂) oxygen tension in the presence of serum‐free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT–PCR. Co‐cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3–1.7‐fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O₂. GAG matrix formation was also enhanced (1.3–1.6‐fold) under 3% O₂ culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG‐rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co‐cultured pellets. However, SOX9 and HIF‐1α mRNA expression were not significantly modulated by co‐culture. Co‐culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co‐delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co‐culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs,human induced pluripotent stem cell‐derived MSCs and human embryonic stem cell‐derived MSCs

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co‐culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co‐cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC‐MSCs) and embryonic stem cells (hESC‐MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary‐like structures were successfully formed on CPC in vitro in all four co‐cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co‐cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co‐cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co‐cultured with hUCMSCs, hiPSC‐MSCs and hESC‐MSCs achieved new bone and vessel density similar to hUVECs co‐cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC‐MSCs and hESC‐MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co‐cultures of hUVECs with hUCMSCs, hiPSC‐MSCs, hESC‐MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co‐culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Mesenchymal progenitor cells derived from traumatized muscle enhance neurite growth

The success of peripheral nerve regeneration is governed by the rate and quality of axon bridging and myelination that occurs across the damaged region. Neurite growth and the migration of Schwann cells is regulated by neurotrophic factors produced as the nerve regenerates, and these processes can be enhanced by mesenchymal stem cells (MSCs), which also produce neurotrophic factors and other factors that improve functional tissue regeneration. Our laboratory has recently identified a population of mesenchymal progenitor cells (MPCs) that can be harvested from traumatized muscle tissue debrided and collected during orthopaedic reconstructive surgery. The objective of this study was to determine whether the traumatized muscle‐derived MPCs exhibit neurotrophic function equivalent to that of bone marrow‐derived MSCs. Similar gene‐ and protein‐level expression of specific neurotrophic factors was observed for both cell types, and we localized neurogenic intracellular cell markers (brain‐derived neurotrophic factor and nestin) to a subpopulation of both MPCs and MSCs. Furthermore, we demonstrated that the MPC‐secreted factors were sufficient to enhance in vitro axon growth and cell migration in a chick embryonic dorsal root ganglia (DRG) model. Finally, DRGs in co‐culture with the MPCs appeared to increase their neurotrophic function via soluble factor communication. Our findings suggest that the neurotrophic function of traumatized muscle‐derived MPCs is substantially equivalent to that of the well‐characterized population of bone marrow‐derived MPCs, and suggest that the MPCs may be further developed as a cellular therapy to promote peripheral nerve regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

Vitamin MK‐7 enhances vitamin D3‐induced osteogenesis in hMSCs: modulation of key effectors in mineralization and vascularization

The osteoblast is the bone‐forming cell and is derived from mesenchymal stem cells (MSCs). Osteo‐inductive substances could represent a useful therapeutic approach during the fracture repair process. The aim of this work was to evaluate the effects of vitamin MK‐7, alone or in association with vitamin D3, in differentiating human MSCs (hMSCs) in vitro along the osteoblastic lineage. In particular, primary endpoints of the study include gene and protein markers of osteoblast differentiation. Considering genes involved in bone formation and mineralization, our data show that vitamin MK‐7 enhances vitamin D3 gene induction of osteocalcin (OC). Among genes related to cell growth and differentiation, a specific effect of vitamin MK‐7 was observed for growth differentiation factor‐10 (GDF10) and insulin‐like growth factor 1 (IGF1), the latter being also involved in the induction of vascular endothelial growth factors (VEGFA). Accordingly, vitamin co‐supplementation greatly affected VEGFA and its receptor fms‐related tyrosine kinase 1 (FLT1), a key factor in both angiogenic and osteogenic processes. These results stress the relevance of MK‐7 and D3 co‐supplementation in the bone‐healing process as able to modulate the expression of genes involved in both mineralization and angiogenesis. Moreover, at the protein level co‐association of vitamins might provide an optimal balance between induction and carboxylation of osteocalcin, essential for its functionality in the extracellular matrix (ECM). Our results may provide hints for therapeutic application of hMSCs in bone disease, clarifying mechanisms involved in stem cell‐mediated bone development, and they also highlight the relevance of co‐supplementation strategies, since single supplementations might result in a suboptimal effect. Copyright © 2012 John Wiley & Sons, Ltd.     

Platelet‐derived growth factor BB gene‐released scaffolds: biosynthesis and characterization

Tissue engineering generally requires three basic elements; stem/progenitor cells, inductive agents and a biomaterial scaffold; the latter is one of the key components which directly influences cellular activity and matrix formation. Commonly used scaffolds to repair defects in general do not induce stem cell recruitment, which is an essential element to tissue regeneration. In this study, fabrication of a scaffold which is capable of restoring damaged tissue through the recruitment of mesenchymal stem cells (MSCs) by gene therapy of the gene encoding platelet‐derived growth factor‐B (PDGF‐B) was investigated. PDGF‐B adenovirus (AdPDGF) was combined into novel mesoporous bioglass–silk fibrin scaffolds, which were characterized for their controlled release and sustained bioactivity. Our results demonstrate that these scaffolds can release PDGF‐B adenovirus for up to 3 weeks and increase MSC recruitment, both in vitro and following subcutaneous implantation in mice. Osseous calvarial defects in mice further demonstrate the ability of these scaffolds to enhance tissue regeneration through stem cell homing. This study demonstrates the potent ability of host stem cells to regenerate tissue defects through recruitment of MSCs via gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.