Functional performance of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold: a pre-clinical in vitro tribological study.

Osteochondral grafts are used for repair of focal osteochondral lesions. Autologous grafts are the gold standard treatment; however, limited graft availability and donor site morbidity restrict use. Therefore, there is a clinical need for different graft sources/materials which replicate natural cartilage function. Chitosan has been proposed for this application. The aim of this study was to assess the biomechanics and biotribology of a bioresorbable chitosan/chitosan-nano-hydroxyapatite osteochondral construct (OCC), implanted in an in vitro porcine knee experimental simulation model. The OCC implanted in different surgical positions (flush, proud and inverted) was compared to predicate grafts in current clinical use and a positive control consisting of a stainless steel graft implanted proud of the cartilage surface. After 3 h (10 800 cycles) wear simulation under a walking gait, subsidence occurred in all OCC samples irrespective of surgical positioning, but with no apparent loss of material and low meniscus wear. Half the predicate grafts exhibited delamination and scratching of the cartilage surfaces. No graft subsidence occurred in the positive controls but wear and deformation of the meniscus were apparent. Implanting a new chitosan-based OCC either optimally (flush), inverted or proud of the cartilage surface resulted in minimal wear, damage and deformation of the meniscus.

Evolution of silicate bioglass particles as porous microspheres with a view towards orthobiologics.

Although FDA approved and clinically utilised, research on 45S5 Bioglass® and S53P4 including other bioactive glasses continues in order to advance their applicability for a range of alternate applications. For example, rendering these particles porous would enable incorporation of varying biological payloads (i.e. cells, drugs and growth factors) and making them spherical would enhance their flow properties enabling delivery to target sites via minimally invasive injection procedures. This paper reports on the manufacture of solid (non-porous; SGMS) and highly porous microspheres (PGMS) with large external pores and fully interconnected porosity from bioactive silicate glass formulations (45S5 and S53P4) via a single stage flame spheroidisation process and their physicochemical properties including in vitro biological response. Morphological and physical characterisation of the SGMS and PGMS revealed interconnected porosity up to 65 ± 5%. Mass loss studies comparing between SGMS and PGMS revealed 1.5 times higher mass loss for the PGMS over 28 days. Also, in vitro bioactivity studies using simulated body fluid (SBF) revealed hydroxyapatite (HA) formation at earlier time point for PGMS compared to their SGMS counterparts (i.e day 1 for PGMS and day 3 for SGMS of 45S5). In addition, HA layers were also formed in cell culture media, with the exception of SGMS of 45S5, which revealed CaP formation with a ratio of 1.52-1.78. Direct cell seeding and indirect cell culture studies (via incubation with microsphere degradation products) revealed mouse 3T3 cells were able to grow and undergo osteogenic differentiation in vitro, confirming cytocompatibility of both 45S5 and S53P4 SGMS and PGMS. More importantly and especially for orthobiologic applications, cells were observed to have migrated within the pores of the PGMS. As such, the PGMS developed from these bioactive silicate glasses are highly promising candidate materials for orthobiologics and alternate applications requiring delivery of biologic payloads.

Effect of varying the Mg with Ca content in highly porous phosphate-based glass microspheres.

This paper reports on the role of phosphate-based glass (PBG) microspheres and their physicochemical properties including in vitro biological response to human mesenchymal stem cells (hMSCs). Solid and porous microspheres were prepared via a flame spheroidisation process. The Mg content in the PBG formulations explored was reduced from 24 to 2 mol% with a subsequent increase in Ca content. A small quantity of TiO2 (1 mol%) was added to the lower Mg-content glass (2 mol%) to avoid crystallisation. Morphological and physical characterisation of porous microspheres revealed interconnected porosity (up to 76 ± 5 %), average external pore sizes of 55 ± 5 µm with surface areas ranging from 0.38 to 0.43 m2 g-1. Degradation and ion release studies conducted compared the solid (non-porous) and porous microspheres and revealed 1.5 to 2.5 times higher degradation rate for porous microspheres. Also, in vitro bioactivity studies using simulated body fluid (SBF) revealed Ca/P ratios for porous microspheres of all three glass formulations were between 0.75 and 0.92 which were within the range suggested for precipitated amorphous calcium phosphate. Direct cell seeding and indirect cell culture studies (via incubation with microsphere degradation products) revealed hMSCs were able to grow and undergo osteogenic differentiation in vitro, confirming cytocompatibility of the formulations tested. However, the higher Mg content (24 mol%) porous microsphere showed the most potent osteogenic response and is therefore considered as a promising candidate for bone repair applications.

Rapid conversion of highly porous borate glass microspheres into hydroxyapatite.

This paper reports on the rapid development of porous hydroxyapatite (HA) microspheres with large external pores and fully interconnected porosity. These porous microspheres were produced by converting borates glasses (namely 45B5, B53P4 and 13-93B) into HA by immersing them in potassium phosphate media and simulated body fluid (SBF). Solid (SGMS) non-porous and highly porous (PGMS) microspheres were prepared from borate glasses via a novel flame spheroidisation process and their physicochemical properties including in vitro biological response were investigated. Morphological and physical characterisation of the PGMS showed interconnected porosity (up to 75 ± 5%) with average external pore sizes of 50 ± 5 µm. Mass loss, ion release, X-ray diffraction (XRD) and Scanning electron microscopy (SEM) analysis confirmed complete conversion to HA in 0.02 M K2HPO4 solution for the PGMS (with exception of 13-93B glass) and at significantly faster rates compared to their SGMS counterparts. However, 13-93B microspheres only converted to HA in Na2HPO4 solution. The in vitro SBF bioactivity studies for all the borate compositions showed HA formation and much earlier for PGMS compared to SGMS. Direct cell culture studies using hMSCs revealed that the converted porous HA microspheres showed enhanced pro-osteogenic properties compared to their unconverted counterparts and such are considered as highly promising candidate materials for bone repair (and orthobiological) applications.

Tailoring Pyro-and Orthophosphate Species to Enhance Stem Cell Adhesion to Phosphate Glasses.

Phosphate-based glasses (PBGs) offer significant therapeutic potential due to their bioactivity, controllable compositions, and degradation rates. Several PBGs have already demonstrated their ability to support direct cell growth and in vivo cytocompatibility for bone repair applications. This study investigated development of PBG formulations with pyro- and orthophosphate species within the glass system (40 - x)P2O5·(16 + x)CaO·20Na2O·24MgO (x = 0, 5, 10 mol%) and their effect on stem cell adhesion properties. Substitution of phosphate for calcium revealed a gradual transition within the glass structure from Q2 to Q0 phosphate species. Human mesenchymal stem cells were cultured directly onto discs made from three PBG compositions. Analysis of cells seeded onto the discs revealed that PBG with higher concentration of pyro- and orthophosphate content (61% Q1 and 39% Q0) supported a 4.3-fold increase in adhered cells compared to glasses with metaphosphate connectivity (49% Q2 and 51% Q1). This study highlights that tuning the composition of PBGs to possess pyro- and orthophosphate species only, enables the possibility to control cell adhesion performance. PBGs with superior cell adhesion profiles represent ideal candidates for biomedical applications, where cell recruitment and support for tissue ingrowth are of critical importance for orthopaedic interventions.

Live Simultaneous Monitoring of Mineral Deposition and Lipid Accumulation in Differentiating Stem Cells.

Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media, modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells.

Porous Phosphate-Based Glass Microspheres Show Biocompatibility, Tissue Infiltration, and Osteogenic Onset in an Ovine Bone Defect Model.

Phosphate-based glasses (PBGs) are bioactive and fully degradable materials with tailorable degradation rates. PBGs can be produced as porous microspheres through a single-step process, using changes in their formulation and geometry to produce varying pore sizes and interconnectivity for use in a range of applications, including biomedical use. Calcium phosphate PBGs have recently been proposed as orthobiologics, based on their in vitro cytocompatibility and ion release profile. In this study, porous microspheres made of two PBG formulations either containing TiO2 (P40Ti) or without (P40) were implanted in vivo in a large animal model of bone defect. The biocompatibility and osteogenic potential of these porous materials were assessed 13 weeks postimplantation in sheep and compared to empty defects and autologous bone grafts used as negative and positive controls. Histological analysis showed marked differences between the two formulations, as lower trabeculae-like interconnection and higher fatty bone marrow content were observed in the faster degrading P40-implanted defects, while the slower degrading P40Ti material promoted dense interconnected tissue. Autologous bone marrow concentrate (BMC) was also incorporated within the P40 and P40Ti microspheres in some defects; however, no significant differences were observed in comparison to microspheres implanted alone. Both formulations induced the formation of a collagen-enriched matrix, from 20 to 40% for P40 and P40Ti2.5 groups, suggesting commitment toward the bone lineage. With the faster degrading P40 formulation, mineralization of the tissue matrix was observed both with and without BMC. Some lymphocyte-like cells and foreign body multinucleated giant cells were observed with P40Ti2.5, suggesting that this more durable formulation might be linked to an inflammatory response. In conclusion, these first in vivo results indicate that PBG microspheres could be useful candidates for bone repair and regenerative medicine strategies and highlight the role of material degradation in the process of tissue formation and maturation.

In vitro cellular testing of strontium/calcium substituted phosphate glass discs and microspheres shows potential for bone regeneration.

Phosphate-based glasses (PBGs) are ideal materials for regenerative medicine strategies because their composition, degradation rates, and ion release profiles can easily be controlled. Strontium has previously been found to simultaneously affect bone resorption and deposition. Therefore, by combining the inherent properties of resorbable PBG and therapeutic activity of strontium, these glasses could be used as a delivery device of therapeutic factors for the treatment of orthopaedic diseases such as osteoporosis. This study shows the cytocompatibility and osteogenic potential of PBGs where CaO is gradually replaced by SrO in the near invert glass system 40P2 O5 ·(16-x)CaO·20Na2 O·24MgO·xSrO (x = 0, 4, 8, 12, and 16 mol%). Direct seeding of MG63 cells onto glass discs showed no significant difference in cell metabolic activity and DNA amount measurement across the different formulations studied. Cell attachment and spreading was confirmed via scanning electron microscopy (SEM) imaging at Days 3 and 14. Alkaline phosphatase (ALP) activity was similarly maintained across the glass compositions. Follow-on studies explored the effect of each glass composition in microsphere conformation (size: 63-125 µm) on human mesenchymal stem cells (hMSCs) in 3D cultures, and analysis of cell metabolic activity and ALP activity showed no significant differences at Day 14 over the compositional range investigated, in line with the observations from MG63 cell culture studies. Environmental SEM and live cell imaging at Day 14 of hMSCs seeded on the microspheres showed cell attachment and colonisation of the microsphere surfaces, confirming these formulations as promising candidates for regenerative medicine strategies addressing compromised musculoskeletal/orthopaedic diseases.

Porous calcium phosphate glass microspheres for orthobiologic applications.

Orthobiologics is a rapidly advancing field utilising cell-based therapies and biomaterials to enable the body to repair and regenerate musculoskeletal tissues. This paper reports on a cost-effective flame spheroidisation process for production of novel porous glass microspheres from calcium phosphate-based glasses to encapsulate and deliver stem cells. Careful selection of the glass and pore-forming agent, along with a manufacturing method with the required processing window enabled the production of porous glass microspheres via a single-stage manufacturing process. The morphological and physical characterisation revealed porous microspheres with tailored surface and interconnected porosity (up to 76 ±â€¯5%) with average pore size of 55 ±â€¯8 µm and surface areas ranging from 0.34 to 0.9 m2 g-1. Furthermore, simple alteration of the processing parameters produced microspheres with alternate unique morphologies, such as with solid cores and surface porosity only. The tuneable porosity enabled control over their surface area, degradation profiles and hence ion release rates. Furthermore, cytocompatibility of the microspheres was assessed using human mesenchymal stem cells via direct cell culture experiments and analysis confirmed that they had migrated to within the centre of the microspheres. The novel microspheres developed have huge potential for tissue engineering and regenerative medicine applications. STATEMENT OF SIGNIFICANCE: This manuscript highlights a simple cost-effective one-step process for manufacturing porous calcium phosphate-based glass microspheres with varying control over surface pores and fully interconnected porosity via a flame spheroidisation process. Moreover, a simple alteration of the processing parameters can produce microspheres which have a solid core with surface pores only. The tuneable porosity enabled control over their surface area, degradation profiles and hence ion release rates. The paper also shows that stem cells not only attach and proliferate but more importantly migrate to within the core of the porous microspheres, highlighting applications for bone tissue engineering and regenerative medicine.

Live Quantitative Monitoring of Mineral Deposition in Stem Cells Using Tetracycline Hydrochloride.

The final stage of in vitro osteogenic differentiation is characterized by the production of mineral deposits containing calcium cations and inorganic phosphates, which populate the extracellular matrix (ECM) surrounding the cell monolayer. Conventional histological techniques for the assessment of mineralization, such as Von Kossa and Alizarin Red S staining, are end point techniques requiring cell fixation. Moreover, in both cases staining quantitation requires dye extraction, which irreversibly alters the ECM conformation and structure, therefore preventing the use of the sample for further analysis. In this study, the use of tetracycline hydrochloride (TC) is proposed for the nondestructive staining, quantitation, and imaging of mineralizing bone-like nodules in live cultures of human bone marrow mesenchymal stem cells cultured under osteogenic conditions. Overnight administration of TC to living cells was shown not to alter the metabolic activity or the progression of cell differentiation. When applied to differentiating cultures, cell exposure to serial doses of TC was found to produce quantifiable fluorescence emission specifically in osteogenic cultures. Incubation with TC enabled fluorescence imaging of mineralized areas in live cultures and the combination with other fluorophores using appropriate filters. These results demonstrate that serial TC administration over the differentiation time course provides a qualitative and quantitative tool for the monitoring and evaluation of the differentiation process in live cells.

Role of substrate biomechanics in controlling (stem) cell fate: Implications in regenerative medicine.

Tissue-specific stem cells reside in a specialized environment known as niche. The niche plays a central role in the regulation of cell behaviour and, through the concerted action of soluble molecules, supportive somatic cells, and extracellular matrix components, directs stem cells to proliferate, differentiate, or remain quiescent. Great efforts have been done to decompose and separately analyse the contribution of these cues in the in vivo environment. Specifically, the mechanical properties of the extracellular matrix influence many aspects of cell behaviour, including self-renewal and differentiation. Deciphering the role of biomechanics could thereby provide important insights to control the stem cells responses in a more effective way with the aim to promote their therapeutic potential. In this review, we provide a wide overview of the effect that the microenvironment stiffness exerts on the control of cell behaviour with a particular focus on the induction of stem cells differentiation. We also describe the process of mechanotransduction and the molecular effectors involved. Finally, we critically discuss the potential involvement of tissue biomechanics in the design of novel tissue engineering strategies.

Substrate stiffness and composition specifically direct differentiation of induced pluripotent stem cells.

Substrate stiffness, biochemical composition, and matrix topography deeply influence cell behavior, guiding motility, proliferation, and differentiation responses. The aim of this work was to determine the effect that the stiffness and protein composition of the underlying substrate has on the differentiation of induced pluripotent stem (iPS) cells and the potential synergy with specific soluble cues. With that purpose, murine iPS-derived embryoid bodies (iPS-EBs) were seeded on fibronectin- or collagen I-coated polyacrylamide (pAA) gels of tunable stiffness (0.6, 14, and 50 kPa) in the presence of basal medium; tissue culture polystyrene plates were employed as control. Specification of iPS cells toward the three germ layers was analyzed, detecting an increase of tissue-specific gene markers in the pAA matrices. Interestingly, soft matrix (0.6 kPa) coated with fibronectin favored differentiation toward cardiac and neural lineages and, in the case of neural differentiation, the effect was potentiated by the addition of specific soluble factors. The generation of mature astrocytes, neural cells, and cardiomyocytes was further proven by immunofluorescence and transmission electron microscopy. In summary, this work emphasizes the importance of using biomimetic matrices to accomplish a more specific and mature differentiation of stem cells for future therapeutic applications.

Neuregulin-1ß induces mature ventricular cardiac differentiation from induced pluripotent stem cells contributing to cardiac tissue repair.

Stem cell-derived cardiomyocytes (CMs) are often electrophysiologically immature and heterogeneous, which represents a major barrier to their in vitro and in vivo application. Therefore, the purpose of this study was to examine whether Neuregulin-1ß (NRG-1ß) treatment could enhance in vitro generation of mature "working-type" CMs from induced pluripotent stem (iPS) cells and assess the regenerative effects of these CMs on cardiac tissue after acute myocardial infarction (AMI). With that purpose, adult mouse fibroblast-derived iPS from α-MHC-GFP mice were derived and differentiated into CMs through NRG-1ß and/or dimethyl sulfoxide (DMSO) treatment. Cardiac specification and maturation of the iPS was analyzed by gene expression array, quantitative real-time polymerase chain reaction, immunofluorescence, electron microscopy, and patch-clamp techniques. In vivo, the iPS-derived CMs or culture medium control were injected into the peri-infarct region of hearts after coronary artery ligation, and functional and histology changes were assessed from 1 to 8 weeks post-transplantation. On differentiation, the iPS displayed early and robust in vitro cardiogenesis, expressing cardiac-specific genes and proteins. More importantly, electrophysiological studies demonstrated that a more mature ventricular-like cardiac phenotype was achieved when cells were treated with NRG-1ß and DMSO compared with DMSO alone. Furthermore, in vivo studies demonstrated that iPS-derived CMs were able to engraft and electromechanically couple to heart tissue, ultimately preserving cardiac function and inducing adequate heart tissue remodeling. In conclusion, we have demonstrated that combined treatment with NRG-1ß and DMSO leads to efficient differentiation of iPS into ventricular-like cardiac cells with a higher degree of maturation, which are capable of preserving cardiac function and tissue viability when transplanted into a mouse model of AMI.