Bone and metal - an orthopaedic perspective on osseointegration of metals

The area of implant osseointegration is of major importance, given the predicted significant rise in the number of orthopaedic procedures and an increasingly ageing population. Osseointegration is a complex process involving a number of distinct mechanisms affected by the implant bulk properties and surface characteristics. Our understanding and ability to modify these mechanisms through alterations in implant design is continuously expanding. The following review considers the main aspects of material and surface alterations in metal implants, and the extent of their subsequent influence on osseointegration. Clinically, osseointegration results in asymptomatic stable durable fixation of orthopaedic implants. The complexity of achieving this outcome through incorporation and balance of contributory factors is highlighted through a clinical case report

Chondrogenic potential of human articular chondrocytes and skeletal stem cells: a comparative study

Regenerative medicine strategies have increasingly focused on skeletal stem cells (SSCs), in response to concerns such as donor site morbidity, dedifferentiation and limited lifespan associated with the use of articular chondrocytes for cartilage repair. The suitability of SSCs for cartilage regeneration, however, remains to be fully determined. This study has examined the chondrogenic potential of human STRO-1-immunoselected SSCs (STRO-1+ SSCs), in comparison to human articular chondrocytes (HACs), by utilising two bioengineering strategies, namely ‘‘scaffold-free’’ three-dimensional(3-D) pellet culture and culture using commercially available, highly porous, 3-D scaffolds with interconnected pore networks. STRO-1+ SSCs were isolated by magnetic-activated cell sorting from bone marrow samples of haematologically normal osteoarthritic individuals following routine hip replacement procedures. Chondrocytes were isolated by sequential enzymatic digestion of deep zone articular cartilage pieces dissected from femoral heads of the same individuals. After expansion in monolayer cultures, the harvested cell populations were centrifuged to form high-density 3-D pellets and also seeded in the 3-D scaffold membranes, followed by culture in serum-free chondrogenic media under static conditions for 21 and 28 days, respectively. Chondrogenic differentiation was determined by gene expression,histological and immunohistochemical analyses. Robust cartilage formation and expression of hyaline cartilage-specific markers were observed in both day-21 pellets and day-28 explants generated using HACs. In comparison, STRO-1+ SSCs demonstrated significantly lower chondrogenic differentiation potential and a tendency for hypertrophic differentiation in day-21 pellets. Culture of STRO-1+ SSCs in the 3-D scaffolds improved the expression of hyaline cartilage-specific markers in day-28 explants, however, was unable to prevent hypertrophic differentiation of the SSC population. The advantages of application of SSCs in tissue engineering are widely recognised; the results of this study, however, highlight the need for further development of cell culture protocols that may otherwise limit the application of this stem cell population in cartilage bioengineering strategies

Epithelial mechanobiology, skin wound healing, and the stem cell niche

AbstractSkin wound healing is a vital process that is important for re-establishing the epithelial barrier following disease or injury. Aberrant or delayed skin wound healing increases the risk of infection, causes patient morbidity, and may lead to the formation of scar tissue. One of the most important events in wound healing is coverage of the wound with a new epithelial layer. This occurs when keratinocytes at the wound periphery divide and migrate to re-populate the wound bed. Many approaches are under investigation to promote and expedite this process, including the topical application of growth factors and the addition of autologous and allogeneic tissue or cell grafts. The mechanical environment of the wound site is also of fundamental importance for the rate and quality of wound healing. It is known that mechanical stress can influence wound healing by affecting the behaviour of cells within the dermis, but it remains unclear how mechanical forces affect the healing epidermis. Tensile forces are known to affect the behaviour of cells within epithelia, however, and the material properties of extracellular matrices, such as substrate stiffness, have been shown to affect the morphology, proliferation, differentiation and migration of many different cell types. In this review we will introduce the structure of the skin and the process of wound healing. We will then discuss the evidence for the effect of tissue mechanics in re-epithelialisation and, in particular, on stem cell behaviour in the wound microenvironment and in intact skin. We will discuss how the elasticity, mechanical heterogeneity and topography of the wound extracellular matrix impact the rate and quality of wound healing, and how we may exploit this knowledge to expedite wound healing and mitigate scarring

Bone tissue engineering

Medical advances have led to a welcome increase in life expectancy. However, accompanying longevity introduces new challenges: increases in age-related diseases and associated reductions in quality of life. The loss of skeletal tissue that can accompany trauma, injury, disease or advancing years can result in significant morbidity and significant socio-economic cost and emphasise the need for new, more reliable skeletal regeneration strategies. To address the unmet need for bone augmentation, tissue engineering and regenerative medicine have come to the fore in recent years with new approaches for de novo skeletal tissue formation. Typically, these approaches seek to harness stem cells, innovative scaffolds and biological factors that promise enhanced and more reliable bone formation strategies to improve the quality of life for many. This review provides an overview of recent developments in bone tissue engineering focusing on skeletal stem cells, vascular development, bone formation and the translation from preclinical in vivo models to clinical delivery

Rapid fabrication and screening of tailored functional 3D biomaterials: Validation in bone tissue repair – Part II

Regenerative medicine strategies place increasingly sophisticated demands on 3D biomaterials to promote tissue formation at sites where tissue would otherwise not form. Ideally, the discovery/fabrication of the 3D scaffolds needs to be high-throughput and uniform to ensure quick and in-depth analysis in order to pinpoint appropriate chemical and mechanical properties of a biomaterial. Herein we present a versatile technique to screen new potential biocompatible acrylate-based 3D scaffolds with the ultimate aim of application in tissue repair. As part of this process, we identified an acrylate-based 3D porous scaffold that promoted cell proliferation followed by accelerated tissue formation, pre-requisites for tissue repair. Scaffolds were fabricated by a facile freeze-casting and an in-situ photo-polymerization route, embracing a high-throughput synthesis, screening and characterization protocol. The current studies demonstrate the dependence of cellular growth and vascularization on the porosity and intrinsic chemical nature of the scaffolds, with tuneable 3D scaffolds generated with large, interconnected pores suitable for cellular growth applied to skeletal reparation. Our studies showed increased cell proliferation, collagen and ALP expression, while chorioallantoic membrane assays indicated biocompatibility and demonstrated the angiogenic nature of the scaffolds. VEGRF2 expression in vivo observed throughout the 3D scaffolds in the absence of growth factor supplementation demonstrates a potential for angiogenesis. This novel platform provides an innovative approach to 3D scanning of synthetic biomaterials for tissue regeneration

Enhancing the osteogenic efficacy of human bone marrow aspirate: concentrating osteoprogenitors using wave-assisted filtration

Background: recent approaches have sought to harness the potential of stem cells to regenerate bone that is lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of osteoprogenitors for such applications. However, previous studies indicated that the concentration of osteoprogenitors present in BMA is less than required for robust bone regeneration. We provide further evidence for the importance of BMA enrichment for skeletal tissue engineering strategies using a novel acoustic wave-facilitated filtration strategy to concentrate BMA for osteoprogenitors, clinically applicable for intraoperative orthopedic use.Methods: femoral BMA from 15 patients of an elderly cohort was concentrated for the nucleated cell fraction against erythrocytes and excess plasma volume via size exclusion filtration facilitated by acoustic agitation. The effect of aspirate concentration was assessed by assays for colony formation, flow cytometry, multilineage differentiation and scaffold seeding efficiency.Results: BMA was filtered to achieve a mean 4.2-fold reduction in volume with a corresponding enrichment of viable and functional osteoprogenitors, indicated by flow cytometry and assays for colony formation. Enhanced osteogenic and chondrogenic differentiation was observed using concentrated aspirate and enhanced cell-seeding efficiency onto allogeneic bone graft as an effect of osteoprogenitor concentration relative specifically to the concentration of erythrocytes in the aspirate.Conclusions: these studies provide evidence for the importance of BMA nucleated cell concentration for both cell differentiation and cell seeding efficiency and demonstrate the potential of this approach for intraoperative application to enhance bone healin

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects

Chondrobags: a high throughput alginate-fibronectin micromass platform for in vitro human cartilage formation

The maintenance and expansion of the cells required for formation of tissue-engineered cartilage has, to date, proven difficult. This is, in part, due to the initial solid phase extracellular matrix demanded by the cells inhabiting this avascular tissue. Herein, we engineer an innovative alginate-fibronectin microfluidic-based carrier construct (termed a chondrobag) equipped with solid phase presentation of growth factors that support skeletal stem cell chondrogenic differentiation while preserving human articular chondrocyte phenotype. Results demonstrate biocompatibility, cell viability, proliferation and tissue-specific differentiation for chondrogenic markers SOX9, COL2A1 and ACAN. Modulation of chondrogenic cell hypertrophy, following culture within chondrobags loaded with TGF-β1, was confirmed by down-regulation of hypertrophic genes COL10A1 and MMP13. MicroRNAs involved in the chondrogenesis process, including miR-140, miR-146b and miR-138 were observed. Results demonstrate the generation of a novel high-throughput, microfluidic-based, scalable carrier that supports human chondrogenesis with significant implications therein for cartilage repair-based therapies

The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering.

Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (?CT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by ?CT analysis (p?<?0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering

Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting.Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting.The ability to create mechanically strong 'cancellous bone-like’ printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair