Cell-matrix Constructs Research Articles

The Angiogenic Potential of Mesenchymal Stem Cells from the Hair Follicle Outer Root Sheath.

Neovascularization is regarded as a pre-requisite in successful tissue grafting of both hard and soft tissues alike. This study considers mesenchymal stem cells from hair follicle outer root sheath (MSCORS) as powerful tools with a neat angiogenic potential that could in the future have wide scopes of neo-angiogenesis and tissue engineering. Autologous MSCORS were obtained ex vivo by non-invasive plucking of hair and they were differentiated in vitro into both endothelial cells and vascular smooth muscle cells (SMCs), two crucial cellular components of vascular grafts. Assessment was carried out by immunostaining, confocal laser-scanning microscopy, gene expression analysis (qRT-PCR), quantitative analysis of anastomotic network parameters, and cumulative length quantification of immunostained α-smooth muscle actin-containing stress fibers (α -SMA). In comparison to adipose mesenchymal stem cells, MSCORS exhibited a significantly higher differentiation efficiency according to key quantitative criteria and their endothelial derivatives demonstrated a higher angiogenic potential. Furthermore, the cells were capable of depositing their own extracellular matrix in vitro in the form of a membrane-cell sheet, serving as a base for viable co-culture of endothelial cells and SMCs integrated with their autologous matrix. Differentiated MSCORS hereby provided a complex autologous cell-matrix construct that demonstrates vascularization capacity and can serve as a base for personalized repair grafting applications.

Mechanotransduction of mitochondrial AMPK and its distinct role in flow-induced breast cancer cell migration

The biophysical microenvironment of the tumor site has significant impact on breast cancer progression and metastasis. The importance of altered mechanotransduction in cancerous tissue has been documented, yet its role in the regulation of cellular metabolism and the potential link between cellular energy and cell migration remain poorly understood. In this study, we investigated the role of mechanotransduction in AMP-activated protein kinase (AMPK) activation in breast cancer cells in response to interstitial fluid flow (IFF). Additionally, we explored the involvement of AMPK in breast cancer cell migration. IFF was applied to the 3D cell-matrix construct. The subcellular signaling activity of Src, FAK, and AMPK was visualized in real-time using fluorescent resonance energy transfer (FRET). We observed that breast cancer cells (MDA-MB-231) are more sensitive to IFF than normal epithelial cells (MCF-10A). AMPK was activated at the mitochondria of MDA-MB-231 cells by IFF, but not in other subcellular compartments (i.e., cytosol, plasma membrane, and nucleus). The inhibition of FAK or Src abolished flow-induced AMPK activation in the mitochondria of MDA-MB-231 cells. We also observed that global AMPK activation reduced MDA-MB-231 cell migration. Interestingly, specific AMPK inhibition in the mitochondria reduced cell migration and blocked flow-induced cell migration. Our results suggest the linkage of FAK/Src and mitochondria-specific AMPK in mechanotransduction and the differential role of AMPK in breast cancer cell migration depending on its subcellular compartment-specific activation.

Hyaluronan Disrupts Cardiomyocyte Organization within 3D Fibrin-Based Hydrogels

The extracellular matrix in vivo contains variable but often large amounts of glycosaminoglycans that influence cell and tissue function. Hyaluronan (HA) is an abundant glycosaminoglycan within the extracellular matrix of the myocardium during early development and in the aftermath of a myocardial infarction. Its flexible anionic structure has a strong influence on mechanical response and interstitial fluid flow within the matrix. Additionally, HA has a direct, biochemical effect on cells through an array of cell-surface receptors, including CD44, RHAMM/CD168, and other surface-exposed structures. Recent studies have shown that HA modulates the response of cardiomyocytes and other cell types to two-dimensional substrates of varying elastic moduli. This study investigates the force response to HA of cardiomyocytes and cardiac fibroblasts within three-dimensional matrices of variable composition and mechanical properties in vitro. HA significantly decreased the force exerted by the cell-matrix constructs in a tensiometer testing platform and within microfabricated tissue gauges. However, its effect was no different from that of alginate, an anionic polysaccharide with the same charge density but no specific transmembrane receptors. Therefore, these results establish that HA exerts a generic physical-chemical effect within three-dimensional hydrogels that must be accounted for when interrogating cell-matrix interactions.

Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices.

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture.

Three-dimensional culture of single embryonic stem-derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling.

In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% βIII-tubulin+ cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES-NSPCs expressed matriptase and secreted MMP-2/9, with MMP-2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES-NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three-dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES-NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin-based hydrogels for ES-NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.

SIS-ECM BIOMATERIAL DECREASES HUMAN CARDIAC MYOFIBROBLAST ACTIVATION AND ATTENUATES LOCAL MATRIX REMODELING

SIS-ECM BIOMATERIAL DECREASES HUMAN CARDIAC MYOFIBROBLAST ACTIVATION AND ATTENUATES LOCAL MATRIX REMODELING

Mechanostimulation changes the catabolic phenotype of human dedifferentiated osteoarthritic chondrocytes.

The treatment of cartilage defects with matrix-embedded autologous chondrocytes is a promising method to support the repair process and to foster reconstitution of full functionality of the joint. Human osteoarthritic chondrocytes were harvest from nine different patients (mean ± SD age 68 ± 8 years) who underwent total knee replacement. The chondrocytes were embedded after a precultivation phase into a collagen I hydrogel. Mid-term intermitted mechanostimulation on matrix-embedded dedifferentiated human osteoarthritic chondrocytes was performed by intermittently applying a cyclic sinusoid compression regime for 4 days (cycles of 1 h of sinusoidal stimulation (1 Hz) and 4 h of break; maximum compression 2.5%). Stimulated (Flex) and non-stimulated (No Flex) cell matrix constructs were analysed concerning the expression of genes involved in tissue metabolism, the content of sulphated glycosaminoglycans (sGAG) and the morphology of the chondrocytes. Gene expression analysis showed a high significant increase in collagen type II expression (p < 0.001), a significant increase in aggrecan expression (p < 0.04) and a high significant decrease in MMP-13 expression (p < 0.001) under stimulation condition compared with unstimulated controls. No significant changes were found in the gene expression rate of MMP-3. This positive effect of the mechanostimulation was confirmed by the analyses of sGAG. Mechanically stimulated cell-matrix constructs had nearly tripled sGAG content than the non-stimulated control (p < 0.002). In addition, histological examination showed that morphology of chondrocytes was altered from a spindle-shaped to a chondrocyte-characteristic rounded phenotype. Mid-term intermitted mechanical stimulation in vitro has the potential to improve the cell quality of cell matrix constructs prepared from dedifferentiated osteoarthritic chondrocytes. This observation may extend the inclusion criteria for matrix-assisted autologous chondrocyte implantation (MACI) and confirms the importance of moderate dynamic compression in clinical rehabilitation after MACI.

Bone marrow derived cell-seeded extracellular matrix: A novel biomaterial in the field of wound management

Aim: Extensive or irreversible damage to the skin often requires additional skin substitutes for reconstruction. Biomaterials have become critical components in the development of effective new medical therapies for wound care. Materials and Methods: In the present study, a cell matrix construct (bone marrow-derived cells (BMdc) seeded extracellular matrix [ECM]) was used as a biological substitute for the repair of full-thickness skin wound. ECM was developed by decellularizing fish swim bladder (FSB). Goat bone marrow-derived cells (G-BMdc) were seeded over this decellularized matrix. Efficacy of this cell matrix construct in wound repair was tested by implanting it over 20 mm2 × 20 mm2 size fullthickness skin wound created over the dorsum of rat. The study was conducted in 16 clinically healthy adult rats of either sex. The animals were randomly divided into 2 equal groups of 8 animals each. In Group I, animal’s wounds were repaired with a cellular FSB matrix. In Group II, wounds were repaired with G-BMdc seeded a cellular FSB matrix. Immune response and efficacy of healing were analyzed. Results: Quality of healing and immuno tolerance to the biological substitute was significantly better in Group II than Group I. Conclusion: Seeding with BMdc increases the wound healing potency and modulates the immune response to a significantly negligible level. The BMdc seeded acellular FSB matrix was found to be a novel biomaterial for wound management.

Toward reconstruction of the subcutaneous fat layer with the use of adipose-derived stromal cell–seeded collagen matrices

Background aimsComplex injuries of the upper and lower extremities often result in scarring and subsequent adhesion formation, which may cause severe pain and distinctly reduce range of motion. In revision surgery, replacement of the missing subcutaneous tissue is desirable to prevent new adhesions, to cushion scarred tendons and nerves and to regain tissue elasticity. Therefore, the objective of this study was the in vitro evaluation of cell-seeded collagen matrices to serve as the basis for the reconstruction of the subcutaneous adipose tissue layer. MethodsFive commercially available acellular dermal collagen matrices were seeded with human adipose-derived stromal cells (hASC). Size and shape stability of cell-matrix constructs were assessed and cell adhesion onto the matrix surface was evaluated histologically. Adipogenic differentiation of hASC on matrices was evaluated by means of histological staining, triglyceride quantification, and quantitative real-time polymerase chain reaction gene expression analysis. ResultsThe collagen matrix Permacol was the only cell-seeded material that exhibited excellent size and shape stability. For Permacol and Strattice, successful seeding with continuous cell layers on top of the matrices was observed. For both matrices, histological staining, triglyceride quantification and messenger RNA expression of adipogenic transcription factors indicated substantial adipogenic differentiation of hASC after long-term induction as well as after short-term induction of only 4 days. ConclusionsOf all matrices investigated, only Permacol exhibited adequate handling stability and the development of a thin adipose tissue layer on top of the matrix. Thus, this matrix appears promising to be used in the development of a subcutaneous cushioning layer after complex injuries involving large scar formation.

Effects of cryopreservation on human mesenchymal stem cells attached to different substrates.

There is a need to preserve cell-seeded scaffolds or cell-matrix constructs for tissue-engineering and other applications. Cryopreservation is likely to be the most practical method. The aim of this study was to investigate how cryopreservation affects cells attached to different substrates and how they respond differently from those in suspension. Human mesenchymal stem cells (hMSCs) were studied for their close relevance to tissue-engineering and stem cell therapy applications, in particular how cryopreservation affects cell adherence, cell growth and the viability of hMSCs attached to different substrates, including glass, gelatin, matrigel and a matrigel sandwich. The effects of cryopreservation on F-actin organization, intracellular pH and mitochondrial localization of the adherent hMSCs were further investigated. It was found that cells attached to a glass surface could hardly survive the common cryopreservation protocol using 10% DMSO and a 1°C/min cooling rate. By contrast, cells attached to gelatin and matrigel could survive to a greater extent. Furthermore, cryopreservation affected the potential of cell attachment and proliferation, resulted in distortion of F-actin, led to alteration of intracellular pH of the hMSCs for all tested substrates and caused a change in the mitochondrial localization of hMSCs on a matrigel substrate and in a matrigel sandwich. Our results showed that cell attachment and cell viability could be improved by changing the interaction between cell and substrate through modification of the substrate properties, which has implications for scaffold design if cell-seeded scaffolds or engineered tissues need to be cryopreserved.

Stem cell-based tissue-engineering for treatment of meniscal tears in the avascular zone

Meniscal tears in the avascular zone have a poor self-healing potential, however partial meniscectomy predisposes the knee for early osteoarthritis. Tissue engineering with mesenchymal stem cells and a hyaluronan collagen based scaffold is a promising approach to repair meniscal tears in the avascular zone. 4 mm longitudinal meniscal tears in the avascular zone of lateral menisci of New Zealand White Rabbits were performed. The defect was left empty, sutured with a 5-0 suture or filled with a hyaluronan/collagen composite matrix without cells, with platelet rich plasma or with autologous mesenchymal stem cells. Matrices with stem cells were in part precultured in chondrogenic medium for 14 days prior to the implantation. Menisci were harvested at 6 and 12 weeks. The developed repair tissue was analyzed macroscopically, histologically and biomechanically. Untreated defects, defects treated with suture alone, with cell-free or with platelet rich plasma seeded implants showed a muted fibrous healing response. The implantation of stem cell-matrix constructs initiated fibrocartilage-like repair tissue, with better integration and biomechanical properties in the precultured stem cell-matrix group. A hyaluronan-collagen based composite scaffold seeded with mesenchymal stem cells is more effective in the repair avascular meniscal tear with stable meniscus-like tissue and to restore the native meniscus.

Is the Transplant Quality at the Time of Surgery Adequate for Matrix-guided Autologous Cartilage Transplantation? A Pilot Study

Matrix-guided autologous chondrocyte transplantation (MACT) has been proposed as an option for treating large full-thickness cartilage defects. However, little is known about the chondrogenic potential of transplants for MACT at the time of implantation, although cell quality and chondrogenic differentiation of the implants are crucial for restoration of function after MACT. We therefore asked: (1) Do MACT implants allow deposition of extracellular cartilage matrix in an in vitro culture model? (2) Are these implants associated with improved knee function 1 year after MACT in large cartilage defects? We retrospectively reviewed all 125 patients with large localized cartilage defects (mean defect size 5 cm(2)) of the knee who were treated with MACT from 2005 to 2010. The mean age was 31 years (range, 16-53 years). Portions of the cell-matrix constructs (n = 50) that were not implanted in the cartilage defects were further cultured and tested for their potential to form articular cartilage. Knee function of all patients was analyzed preoperatively, 3 months, and 1 year postoperatively with the International Knee Documentation Committee (IKDC) score. In vitro assessment of the cell-matrix implants showed chondrogenic differentiation with positive staining for glycosaminoglycans and collagen II in all cultures. Enzyme-linked immunosorbent assay showed an increase of collagen II production. We observed an improvement in median IKDC score from 41 to 67 points at last followup. Cartilage extracellular matrix deposition shows adequate implant quality for MACT at the time of implantation and justifies the use for treatment of large cartilage defects.

Mesenchymal Stem Cell Chondrogenesis: Composite Growth Factor–bioreactor Synergism for Human Stem Cell Chondrogenesis

Effective mesenchymal stem cell chondrogenesis can be accomplished by using a tailored mechanical-biochemical stimulus. To achieve this requires parallel suppression of hypertrophy and osteogenesis. We compared the effects of isolated bioreactor stimulation, isolated growth factor (TGF-β1 or IGF-1) application and their combined stimulation on human bone marrow-derived mesenchymal stem cells cultured within 3D scaffolds. Free-swelling cell-matrix constructs underwent identical growth factor stimulation for control. Mechanical stimulation provoked stronger chondrogenic differentiation than free-swelling culture. Chondrogenesis by the addition of TGF-β1 was enhanced compared with single physical stimulation. There were no such effects under the influence of IGF-1 alone. Composite application of multiaxial mechanical stimulation plus TGF-β1 and IGF-1 not only triggered the strongest chondrogenesis overall, but also the strongest hypertrophy and osteogenesis. Bioreactor-induced chondrogenic differentiation of human mesenchymal stem cells can be effectively enhanced by growth factor addition, while the partially effective suppression of unwanted signs of endochondral ossification requires further scientific input.

Cell mediated contraction in 3D cell-matrix constructs leads to spatially regulated osteogenic differentiation

During embryonic development, morphogenetic processes give rise to a variety of shapes and patterns that lead to functional tissues and organs. While the impact of chemical signals on these processes is widely studied, the role of physical cues is less understood. The aim of this study was to test the hypothesis that the interplay of cell mediated contraction and mechanical boundary conditions alone can result in spatially regulated differentiation in simple 3D constructs. An experimental model consisting of a 3D cell-gel construct and a finite element (FE) model were used to study the effect of cellular traction exerted by mesenchymal stem cells (MSCs) on an initially homogeneous matrix under inhomogeneous boundary conditions. A robust shape change is observed due to contraction under time-varying mechanical boundary conditions, which is explained by the finite element model. Furthermore, distinct local differences in osteogenic differentiation are observed, with a spatial pattern independent of osteogenic factors in the culture medium. Regions that are predicted to have experienced relatively high shear stress at any time during contraction correlate with the regions of distinct osteogenesis. Taken together, these results support the underlying hypothesis that cellular contractility and mechanical boundary conditions alone can result in spatially regulated differentiation. These results will have important implications for tissue engineering and regeneration.

The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration

Matrix-assisted chondrocyte transplantation (m-ACI) still lacks any standardization in its execution in terms of cell passage (P), cell yield (C) and in vitro membrane-holding time (T). It was the goal of this study to analyze the effect of shifting cell culture parameters (P, C, T) on the in vitro as well as in vivo effort of a regulated animal m-ACI. Autologous rabbit knee articular chondrocytes were seeded within bilayer collagen I/III 3-D matrices in variation of P, C and T. Each time, 2 PCT-identical by 2 PCT-identical cell-matrix-constructs (CMC)/animal were created. Simultaneously 2 (PCT-distinct) were re-implanted (CMC-e) autologous into artificial trochlear pristine chondral defects in vivo to remain for 12 weeks while the remaining 2 were harvested (CMC-i) for immediate in vitro analysis at the time of transplantation of their identical twins. mRNA of both, CMC-e regenerates and CMC-i membranes, was analyzed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 expression by use of a mixed linear model, multiple regression analysis. Generally, CMC-i values were higher than CMC-e values for differentiation targets; the opposite was true for dedifferentiation targets. Regarding individual gene expression, in vivo regenerate cell-matrix properties were significantly dependent on initial cell-matrix in vitro values as a sign of linearity. The parameter membrane-holding time (T) had strongest effects on the resulting mRNA expression with slightly less impact of the parameter passage (P), whereas cell yield (C) had clearly less effects. Noting differences between in vitro and in vivo data, in general, optimal expression patterns concerning chondrogenic differentiation were achieved by few passages, medium cellular yield, short membrane-holding time. Clinical m-ACI may benefit from optimal orchestration of the cell culture parameters passage, yield and time.

Effect of substrate composition and alignment on corneal cell phenotype.

Corneal blindness is a significant problem treated primarily by corneal transplants. Donor tissue supply is low, creating a growing need for an alternative. A tissue-engineered cornea made from patient-derived cells and biopolymer scaffold materials would be widely accessible to all patients and would alleviate the need for donor sources. Previous work in this lab led to a method for electrospinning type I collagen scaffolds for culturing corneal fibroblasts ex vivo that mimics the microenvironment in the native cornea. This electrospun scaffold is composed of small-diameter, aligned collagen fibers. In this study, we investigate the effect of scaffold nanostructure and composition on the phenotype of corneal stromal cells. Rabbit-derived corneal fibroblasts were cultured on aligned and unaligned collagen type I fibers ranging from 50 to 300 nm in diameter and assessed for expression of α-smooth muscle actin, a protein marker upregulated in hazy corneas. In addition, the optical properties of the cell-matrix constructs were assessed using optical coherence microscopy. Cells grown on collagen scaffolds had reduced myofibroblast phenotype expression compared to cells grown on tissue culture plates. Cells grown on aligned collagen type I fibers downregulated α-smooth muscle actin protein expression significantly more than unaligned collagen scaffolds, and also exhibited reduced overall light scattering by the tissue construct. These results suggest that aligned collagen type I fibrous scaffolds are viable platforms for engineering corneal replacement tissue.

Role of mesenchymal stem cells in tissue engineering of meniscus

Tissue engineering is a promising approach for the treatment of tissue defects. Mesenchymal stem cells are of potential use as a source of repair cells or of important growth factors for tissue engineering. The purpose of this study was to examine the role of mesenchymal stem cells in meniscal tissue repair. This was tested using several cell and biomaterial-based treatment options for repair of defects in the avascular zone of rabbit menisci. Circular meniscal punch defects (2 mm) were created in the avascular zone of rabbit menisci and left empty or filled with hyaluronan-collagen composite matrices without cells, loaded with platelet-rich plasma, autologous bone marrow, or autologous mesenchymal stem cells. In some experiments, matrices with stem cells were precultured in chondrogenic medium for 14 days before implantation. Rabbits were then allowed free cage movement after surgery for up to 12 weeks. Untreated defects and defects treated with cell-free implants had muted fibrous healing responses. Neither bone marrow nor platelet-rich plasma loaded in matrices produced improvement in healing compared with cell-free implants. The implantation of 14 days precultured chondrogenic stem cell-matrix constructs resulted in fibrocartilage-like repair tissue, which was only partially integrated with the native meniscus. Non-precultured mesenchymal stem cells in hyaluronan-collagen composite matrices stimulated the development of completely integrated meniscus-like repair tissue. The study shows the necessity of mesenchymal stem cells for the repair of meniscal defects in the avascular zone. Mesenchymal stem cells seem to fulfill additional repair qualities besides the delivery of growth factors.

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and/or increasing insulin on the stratification and secretion of aligned matrix by fourth- to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin-transferrin-selenium were investigated. High-resolution differential interference contrast microscopy, quick-freeze/deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell-matrix constructs. In a medium containing 1% each of serum and insulin-transferrin-selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell-matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils.

Bone Regeneration Using an Acellular Extracellular Matrix and Bone Marrow Mesenchymal Stem Cells Expressing Cbfa1

To treat bone defects, tissue-engineering methods combine an appropriate scaffold with cells and osteogenic signals to stimulate bone repair. Mesenchymal stem cells (MSCs) derived from adult bone marrow are an ideal source of cells for tissue engineering, in particular for applications in skeletal and hard tissue repair. Core binding factor alpha1 (Cbfa1) is an essential transcription factor for osteoblast differentiation. However, the effects of Cbfa1 on MSCs in vitro and in vivo have not been well characterized. In this study, we found that MSCs modified genetically to express Cbfa1 promoted the healing of segmental defects of the radius in rabbits. First, osteogenic differentiation of MSCs transfected with an adenovirus encoding Cbfa1 was demonstrated. Expression of mRNA from a number of osteoblastic marker genes, including osteocalcin, osteopontin, and type I collagen, was detected. In addition, alkaline phosphatase activity and increased osteocalcin content were observed. The cells expressing the Cbfa1 gene were then combined with acellular bone extracellular matrix in a flow perfusion culture system. Finally, the cell-matrix constructs were implanted into radius defects in the rabbit model. After 12 weeks, radiographic, histological, and biomechanical analyses showed that MSCs modified with the Cbfa1 gene resulted in a significantly higher amount of newly-formed bone and rebuilding of the marrow cavity than control cell-matrix constructs. This study indicates that MSCs modified with the Cbfa1 gene can act as suitable seed cells for the regeneration of bone defects.

Cultivation of human bone marrow stromal cells on three-dimensional scaffolds of mineralized collagen: influence of seeding density on colonization, proliferation and osteogenic differentiation

In this study human bone marrow stromal cells (hBMSCs) were cultured on three-dimensional porous scaffolds of biomimetically mineralized collagen type I developed for bone engineering. Three different cell numbers were used for seeding of the nanocomposites, and the impact of the seeding density on proliferation and osteogenic differentiation of hBMSCs was investigated. In addition, the effect of the seeding cell number on seeding efficiency and distribution of the cells within the scaffolds was studied. Our data revealed that the open and interconnecting porosity of the mineralized collagen scaffolds allows a very efficient seeding for all seeding densities tested. Although penetration of the cells into the interior of the scaffolds was demonstrated for all seeding densities, the application of higher cell numbers resulted in a better colonization also of the deeper scaffold regions. A substantial influence of the seeding density was observed on proliferation and osteogenic differentiation of hBMSCs. Thus, the highest proliferation rate and specific alkaline phosphatase activity was found for the cell matrix constructs seeded with the lowest density. RT-PCR analyses revealed a higher expression of alkaline phosphatase and bone sialoprotein II at lower seeding densities; however, expression of osteopontin was unaffected by the seeding cell number. Our results demonstrated that the seeding density might be an important factor for the development of optimal cell matrix constructs for bone tissue engineering.