Sub-confluent culture of human mesenchymal stromal cells on biodegradable polycaprolactone microcarriers enhances bone healing of rat calvarial defect.

In the current emerging trend of using human mesenchymal stromal cell (MSCs) for cell therapy, large quantities of cells are needed for clinical testing. Current methods of culturing cells, using tissue culture flasks or cell multilayer vessels, are proving to be ineffective in terms of cost, space and manpower. Therefore, alternatives such as large-scale industrialized production of MSCs in stirred tank bioreactors using microcarriers (MCs) are needed. Moreover, the development of biodegradable MCs for MSC expansion can streamline the bioprocess by eliminating the need for enzymatic cell harvesting and scaffold seeding for bone-healing therapies. Our previous studies described a process of making regulated density (1.06 g/cm3) porous polycaprolactone biodegradable MCs Light Polycarprolactone (LPCL) (MCs), which were used for expanding MSCs from various sources in stirred suspension culture. Here, we use human early MSCs (heMSCs) expanded on LPCL MCs for evaluation of their osteogenic differentiation potential in vitro as well as their use in vivo calvarial defect treatment in a rat model. In summary, (i) in vitro data show that LPCL MCs can be used to efficiently expand heMSCs in stirred cultures while maintaining surface marker expression; (ii) LPCL MCs can be used as scaffolds for cell transfer for transplantation in vivo; (iii) 50% sub-confluency, mid-logarithmic phase, on LPCL MCs (50% confluent) exhibited higher secretion levels of six cytokines (interleukin [IL]-6, IL-8, Vascular endothelial growth factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), growth-regulated oncogene-α (GRO-α) and stromal cell-derived factor-1α (SDF-1α)) as compared with 100% confluent, stationary phase cultures (100% confluent); (iv) these 50% confluent cultures demonstrated better in vitro osteogenic differentiation capacity as compared with 100% confluent cultures (higher levels of calcium deposition and at earlier stage); the improved bone differentiation capacity of these 50% confluent cultures was also demonstrated at the molecular level by higher expression of early osteoblast genes Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), collagen type I, osterix and osteocalcin); and (v) in vivo implantation of biodegradable LPCL MCs covered with 50% heMSCs into rats with calvarial defect demonstrated significantly better bone formation as compared with heMSCs obtained from monolayer cultures (5.1 ± 1.6 mm3 versus 1.3 ± 0.7 mm3). Moreover, the LPCL MCs covered with 50% heMSCs supported better in vivo bone formation compared with 100% confluent culture (2.1 ± 1.3 mm3). Taken together, our study highlights the potential of implanting 50% confluent MSCs propagated on LPCL MCs as optimal for bone regeneration. This methodology allows for the production of large numbers of MSCs in a three-dimensional (3D) stirred reactor, while supporting improved bone healing and eliminating the need for a 3D matrix support scaffold, as traditionally used in bone-healing treatments.

Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials.

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

Biodegradable ECM-coated PCL microcarriers support scalable human early MSC expansion and in vivo bone formation.

BACKGROUND AIMS: Human mesenchymal stromal cells or marrow stromal cells (MSCs) are of great interest for bone healing due to their multi-potency and trophic effects. However, traditional MSC expansion methods using 2-dimensional monolayer (MNL) flasks or cell stacks are limited by labor-intensive handling, lack of scalability, the need for enzymatic cell harvesting and the need for attachment to a scaffold before in vivo delivery. Here, we present a biodegradable microcarrier and MSC bioprocessing system that may overcome the abovementioned challenges. METHODS: We cultured human early MSCs (heMSCs) on biodegradable polycaprolactone microcarriers (PCL MCs) coated with extracellular matrix (ECM) and evaluated the in vitro osteogenic differentiation and in vivo bone formation capacity of ECM-coated PCL MC-bound heMSCs compared with conventional MNL-cultured cells. RESULTS: We found that heMSCs proliferate well on PCL MCs coated with a fibronectin, poly-l-lysine, and fibronectin (FN+PLL+FN) coating (cPCL MCs). During in vitro osteogenic induction, heMSCs cultured on cPCL MCs displayed a 68% increase in specific calcium deposition compared with cultures on MNL. In a mouse ectopic mineralization model, bone mass was equivalent for MNL-expanded and cPCL MC-bound heMSC implants but higher in both cases when compared with cell-free cPCL MC implants at 16 weeks post-implantation. In summary, compared with MNL cultures, biodegradable MC MSC cultures provide the benefits of large-scale expansion of cells and can be delivered in vivo, thereby eliminating the need for cell harvesting and use of scaffolds for cell delivery. These results highlight the promise of delivering heMSCs cultured on cPCL MCs for bone applications.

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin ß1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers.

BACKGROUND: Mesenchymal stem cells (MSCs) are of great interest in bone regenerative medicine due to their osteogenic potential and trophic effects. However, challenges to large-scale production of MSCs can hinder the translation of MSC therapies. 3D Microcarrier (MC)-based MSC culture presents a scalable and cost-effective alternative to conventional methods of expansion in 2D monolayers. Furthermore, biodegradable MCs may allow for MC-bound MSC delivery without enzymatic harvest for selected applications such as bone healing. However, the effects of cell expansion on microcarriers and enzymatic cell harvest on MSC phenotype and osteogenic differential potential are not well understood. In this study, we characterized human fetal MSCs (hfMSCs) after expansion in 3D microcarrier spinner or 2D monolayer cultures. Following expansion, we compared osteogenic differentiation of cultures seeded with 3D MC-harvested, 3D MC-bound and conventional 2D monolayer (MNL)-harvested cells when cultured in osteogenic induction media on collagen-coated plates. RESULTS: Fetal MSCs expanded on both 3D agitated Microcarriers (MC) and 2D Plastic static monolayer (MNL) cultures express high levels of MSC surface markers. MC-harvested hfMSCs displayed higher expression of early osteogenic genes but slower mineralization kinetics compared to MNL-harvested MSCs during osteogenic induction. However, in the comparison between MC-bound and MC-harvested hfMSCs, osteogenic genes were upregulated and mineralization kinetics was accelerated in the former condition. Importantly, 3D MC-bound hfMSCs expressed higher levels of osteogenic genes and displayed either higher or equivalent levels of mineralization, depending on the cell line, compared to the classical monolayer cultures use in the literature (MNL-harvested hfMSCs). CONCLUSION: Beyond the processing and scalability advantages of the microcarrier culture, hfMSCs attached to MCs undergo robust osteogenic differentiation and mineralization compared to enzymatically harvested cells. Thus biodegradable/biocompatible MCs which can potentially be used for cell expansion as well as a scaffold for direct in vivo delivery of cells may have advantages over the current methods of monolayer-expansion and delivery post-harvest for bone regeneration applications.

Nanoscale engineering of extracellular matrix-mimetic bioadhesive surfaces and implants for tissue engineering.

BACKGROUND: The goal of tissue engineering is to restore tissue function using biomimetic scaffolds which direct desired cell fates such as attachment, proliferation and differentiation. Cell behavior in vivo is determined by a complex interaction of cells with extracellular biosignals, many of which exist on a nanoscale. Therefore, recent efforts in tissue engineering biomaterial development have focused on incorporating extracellular matrix- (ECM) derived peptides or proteins into biomaterials in order to mimic natural ECM. Concurrent advances in nanotechnology have also made it possible to manipulate protein and peptide presentation on surfaces on a nanoscale level. SCOPE OF REVIEW: This review discusses protein and peptide nanopatterning techniques and examples of how nanoscale engineering of bioadhesive materials may enhance outcomes for regenerative medicine. MAJOR CONCLUSIONS: Synergy between ECM-mimetic tissue engineering and nanotechnology fields can be found in three major strategies: (1) Mimicking nanoscale orientation of ECM peptide domains to maintain native bioactivity, (2) Presenting adhesive peptides at unnaturally high densities, and (3) Engineering multivalent ECM-derived peptide constructs. GENERAL SIGNIFICANCE: Combining bioadhesion and nanopatterning technologies to allow nanoscale control of adhesive motifs on the cell-material interface may result in exciting advances in tissue engineering. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype.

Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The ß1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete ß1 integrin knockout results in embryonic lethality, studies of ß1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that ß1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of ß1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of ß1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and osteocalcin-Cre lines to generate conditional ß1 integrin deletions, where Cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific ß1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific ß1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of ß1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that ß1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while ß1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic chondryocyte-lineage cells regulate incisor eruption and perinatal bone formation in both intramembranously and endochondrally formed bones in young, rapidly growing mice. In contrast, the osteocalcin-specific ß1 integrin deletion had only minor effects on skeletal phenotype.

Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle.

Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2ß1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.

Extracellular matrix-mimetic adhesive biomaterials for bone repair.

Limited osseointegration of current orthopedic biomaterials contributes to the failure of implants such as arthroplasties, bone screws, and bone grafts, which present a large socioeconomic cost within the United States. These implant failures underscore the need for biomimetic approaches that modulate host cell-implant material responses to enhance implant osseointegration and bone formation. Bioinspired strategies have included functionalizing implants with extracellular matrix (ECM) proteins or ECM-derived peptides or protein fragments, which engage integrins and direct osteoblast adhesion and differentiation. This review discusses (1) bone ECM composition and key integrins implicated in osteogenic differentiation, (2) the use of implants functionalized with ECM-mimetic peptides/protein fragments, and (3) growth factor-derived peptides to promote the mechanical fixation of implants to bone and to enhance bone healing within large defects.

Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair.

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the alpha(2)beta(1) integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.

A thixotropic nanocomposite gel for three-dimensional cell culture.

Thixotropic materials, which become less viscous under stress and return to their original state when stress is removed, have been used to deliver gel-cell constructs and therapeutic agents. Here we show that a polymer-silica nanocomposite thixotropic gel can be used as a three-dimensional cell culture material. The gel liquefies when vortexed--allowing cells and biological components to be added--and resolidifies to trap the components when the shear force from spinning is removed. Good permeability of nutrients and gases through the gel allows various cell types to proliferate and be viable for up to three weeks. Human mesenchymal stem cells cultured in stiffer gels developed bone-like behaviour, showing that the rheological properties of the gel can control cell differentiation. No enzymatic, chemical, or photo-crosslinking, changes in ionic strength or temperature are required to form or liquefy the gel, offering a way to sub-culture cells without using trypsin-a protease commonly used in traditional cell culture techniques.