Elena García-Gareta

A Novel Multiparameter In Vitro Model of Three-Dimensional Cell Ingress Into Scaffolds for Dermal Reconstruction to Predict In Vivo Outcome

Abstract The clinical demand for effective dermal substitutes continues as current commercially available products present limitations. However, there are no definitive in vitro methods to predict in vivo outcomes such as integration, cellularization and contraction, which may help the development of new dermal scaffolds. This study aimed to develop a multiparameter in vitro model of three-dimensional (3D) cell ingress into dermal scaffolds to predict in vivo outcomes of new dermal scaffolds under development. A new dermal scaffold, Smart Matrix, was compared to the scar-forming contractile collagen gel model and to the clinically well-established Integra® and Matriderm®. Parameters studied were cell viability and proliferation, apoptosis, matrix contraction, cell morphology, α-smooth muscle actin, and growth factor expression. Combinatorial evaluation of the results in a scoring matrix showed that Smart Matrix could offer an advantage over existing products. This method would be useful as an international golden scoring matrix to develop new dermal scaffolds that effectively improve the existing products, thus enabling better treatments for burns or chronic wounds.

Bone remodelling in vitro: Where are we headed?: -A review on the current understanding of physiological bone remodelling and inflammation and the strategies for testing biomaterials in vitro

Bone remodelling is a dynamic process required for the maintenance of bone architecture in response to the changing mechanical needs. It is also a vital process during the repair of bone tissue following injury. Clinical intervention in terms of autografting or allografting is often required to heal bone injuries where physiological healing fails. The use of biomaterials as alternatives to autografts and allografts has spurred a significant research interest into further development of biomaterials for better clinical outcomes. Unfortunately, many biomaterials fail to make it to the clinic or fail after implantation due to the inconsistencies observed between in vitro and in vivo studies. It is therefore important to mimic the in vivo situation as closely as possible in an in vitro setting for testing biomaterials. The current in vitro models focus mostly on investigating the behaviour of osteoblast progenitors with the biomaterial under development as well as assessing the behaviour of osteoclasts, endothelial cells etc. However, the sequence of events that take place during bone healing or remodelling are not incorporated into the current in vitro models. This review highlights our current understanding of the physiological bone remodelling and the bone healing process followed by strategies to incorporate both the physiological and pathophysiological events into an in vitro environment. Here, we propose three strategies for the assessment of biomaterials for bone, which includes; (1) testing biomaterials in the presence of immune cells, (2) testing biomaterials for osteogenesis, and (3) testing biomaterials in the presence of osteoclasts followed by osteoblasts to recapitulate the physiological events of bone resorption prior to bone formation. The focus of this review is to discuss the third strategy in details as the first two strategies are currently incorporated into a majority of in vitro experiments.

An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds.

The use of biopolymers as a three dimensional (3D) support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface properties to assess whether seeding efficiencies and cell growth on 3D scaffolds were affected. Cell attachment and growth of murine calvarial osteoblast (MC3T3-E1) cells within a melt-electrospun poly-ε-caprolactone scaffold were assessed when cultured in either “low-adhesive” non-treated or corona discharged-treated well-plates. Increased cell adhesion was observed on the scaffold placed in the surface treated culture plates compared to the scaffold in the non-treated plates 24 h after seeding, although it was not significant. However, higher cell metabolic activity was observed on the bases of all well-plates than on the scaffold, except for day 21, well metabolic activity was higher in the scaffold contained in non-treated plate than the base. These results indicate that there is no advantage in using non-treated plates to improve initial cell seeding in 3D polymeric tissue engineering scaffolds, however non-treated plates may provide an improved metabolic environment for long-term studies.

Collagen gels and the ‘Bornstein legacy’: from a substrate for tissue culture to cell culture systems and biomaterials for tissue regeneration

As collagen is the main structural component of connective tissues and skin, much effort was made in the past and still today to use it in cell culture applications. Moreover, collagen biomaterials are widely used in tissue regeneration, including the treatment of burns and chronic wounds. The great implications of the research carried out by Bornstein, Ehrmann and Gey on collagen preparations in the 1950s for cell culture and more recently tissue engineering and regeneration are described in this commentary. Specifically, it is explored why the 1958 paper on ‘Reconstituted Rat‐Tail Collagen Used as Substrate for Tissue Cultures on Coverslips in Maximow Slides and Roller Tubes’ by M. B. Bornstein has made an invaluable contribution to the field.

Method for estimating protein binding capacity of polymeric systems

Composite biomaterials made from synthetic and protein-based polymers are extensively researched in tissue engineering. To successfully fabricate a protein-polymer composite, it is critical to understand how strongly the protein binds to the synthetic polymer, which occurs through protein adsorption. Currently, there is no cost-effective and simple method for characterizing this interfacial binding. To characterize this interfacial binding, we introduce a simple three-step method that involves: 1) synthetic polymer surface characterisation, 2) a quick, inexpensive and robust novel immuno-based assay that uses protein extraction compounds to characterize protein binding strength followed by 3) an in vitro 2D model of cell culture to confirm the results of the immuno-based assay. Fibrinogen, precursor of fibrin, was adsorbed (test protein) on three different polymeric surfaces: silicone, poly(acrylic acid)-coated silicone and poly(allylamine)-coated silicone. Polystyrene surface was used as a reference. Characterisation of the different surfaces revealed different chemistry and roughness. The novel immuno-based assay showed significantly stronger binding of fibrinogen to both poly(acrylic acid) and poly(allylamine) coated silicone. Finally, cell studies showed that the strength of the interaction between the protein and the polymer had an effect on cell growth. This novel immuno-based assay is a valuable tool in developing composite biomaterials of synthetic and protein-based polymers with the potential to be applied in other fields of research where protein adsorption onto surfaces plays an important role.

Biomimetic surface functionalization of clinically relevant metals used as orthopaedic and dental implants

Titanium and its alloys or tantalum (Ta) are materials used in orthopaedic and dental implants due to their excellent mechanical properties and biocompatibility. However, their bioactivity and osteoconductivity is low. With a view to improving the bioactivity of these materials we hypothesised that the surface of Ta and TiAl6V4 can be functionalised with biomimetic, amorphous nano-sized calcium phosphate (CaP) apatite-like deposits, instead of creating uniform coatings, which can lead to flaking, delamination and poor adherence. We used Ta and TiAl6V4 metal discs with smooth and rough surfaces. Amorphous CaP apatite-like particles were deposited on the different surfaces by a biomimetic rapid two-step soaking method using concentrated simulated body fluid (SBF) solutions without a pre-treatment of the metal surfaces to induce CaP deposition. Immersion times in the second SBF solution of 48 and 18 h for Ta and TiAl6V4 respectively produced CaP deposits composed of amorphous globular nano-sized particles that also contained Mg, C and O. Longer immersion times produced more uniform coatings as well as an undesired calcite mineral phase. Prediction of in vivo behaviour by immersion in regular SBF showed that the obtained CaP deposits would act as a catalyst to rapidly form a Ca deficient CaP layer that also incorporates Mg. The amorphous CaP apatite-like deposits promoted initial attachment, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells. Finally, we used our method to functionalise 3D porous structures of titanium alloy made by selective laser sintering. Our study uses a novel and cost-effective approach to functionalise clinically relevant metal surfaces in order to increase the bioactivity of these materials, which could improve their clinical performance.

Kerr's coining of 'Apoptosis' and its relevance in skin wound healing and fibrosis

‘Apoptosis’, a Greek word meaning ‘dropping off’ or ‘falling off’, was first used by Kerr and team to label a form of cell death (Table 1) by a controlled genetic process whose morphological features were described in their seminal paper Kerr et al. (1), commented in this article. (The full article is freely available in PubMed Central http://www.ncbi.nlm.nih.gov/pubmed/4561027). Although they were the first to coin the term, it had been observed by a handful of scientists since the mid-nineteenth century and described under names such as ‘necrobiosis’ and ‘chromatolysis’ (2). By mid-1900’s, spontaneous cell death was a concept known to developmental biologists, but the features involved were not elucidated, and it drew little interest from the wider scientific community (2). It was not until Kerr’s landmark paper that ‘apoptosis’ as we know it came to be recognized as a phenomenon distinctly different from ‘necrosis’. Kerr himself initially used the term ‘shrinkage necrosis’ to describe his observations of cell death. In his 1965 experiment, he ligated a branch of the hepatic portal vein resulting in liver atrophy. He observed classical necrosis and a different form of cell death in the surviving tissue as liver shrinkage occurred. Some hepatocytes rounded off into small bodies of cytoplasm which sometimes contained condensed chromatin, these were then engulfed by the neighbouring cells or by specialized histiocytes (3). Kerr observed that these cells were in reality dying off to probably achieve a balance between surviving cells and the remaining blood supply (4). In the following years, he began to study the morphological features of ‘shrinkage necrosis’ using the electron microscope. On a sabbatical leave to Aberdeen, he collaborated with pathologists Alastair Currie and Andrew Wyllie who had themselves observed ‘shrinkage necrosis’ in rat adrenal cortices when adrenocorticotropic hormone was suppressed (3). It was here that they coined the term ‘apoptosis’ and described its morphological features. Decades later, this has evolved into the current understanding of the morphological hallmarks of apoptosis summarized in Fig. 1. Kerr et al. studied various instances of spontaneous cell loss such as in neoplasms, types of liver and adrenal injury, and in ontogenesis. They came to the fascinating conclusion that the morphological and ultra structural features exhibited by the phenomenon of cell death in each case were the same (1). We believe this was a remarkable achievement as nobody so far had realized that the features of this process in various tissues was similar enough to propose it as a concept of programmed cell death which as Kerr put it ‘plays a complementary but opposite role to mitosis’ in maintaining tissue homoeostasis. Interestingly, Kerr’s paper on apoptosis did not lead to an immediate flurry of research on the topic. However, the invention of novel techniques to detect apoptosis in cells, the discovery of genes regulating apoptosis in Caenorhabditis elegans and their mammalian homologues such as the BCL2 gene all played crucial roles in kindling scientific interest towards elucidating the biochemical mechanisms of apoptosis and the apoptotic machinery (5). It is now a well-known fact that altered tissue homoeostasis as a result of too much or too less apoptosis leads to a number of cutaneous pathologies such as toxic epidermal necrolysis, psoriasis, forms of skin cancer and fibrosis (6). Moreover, the molecular

Apoptotic primary normal human dermal fibroblasts for in vitro models of fibrosis.

Recent studies show that apoptosis affects surrounding tissue, playing a role in diseases such as fibrosis, a significant global disease burden. Elucidating the mechanisms by which the different apoptotic cells present during fibrotic wound healing affect their environment would enable development of new therapies. We describe here a simple, rapid, and cost-effective method for inducing apoptosis of primary normal human dermal fibroblasts without affecting the overall cell viability of the population. Such population could be used for in vitro models of fibrotic wound healing in co-culture with other cells involved in this process to study events such as apoptosis-induced proliferation.

The importance of factorial design in tissue engineering and biomaterials science: Optimisation of cell seeding efficiency on dermal scaffolds as a case study:

This article presents a case study to show the usefulness and importance of using factorial design in tissue engineering and biomaterials science. We used a full factorial experimental design (2 × 2 × 2 × 3) to solve a routine query in every biomaterial research project: the optimisation of cell seeding efficiency for pre-clinical in vitro cell studies, the importance of which is often overlooked. In addition, tissue-engineered scaffolds can be cellularised with relevant cell type(s) to form implantable tissue constructs, where the cell seeding method must be reliable and robust. Our results show the complex relationship between cells and scaffolds and suggest that the optimum seeding conditions for each material may be different due to different material properties, and therefore, should be investigated for individual scaffolds. Our factorial experimental design can be easily translated to other cell types and three-dimensional biomaterials, where multiple interacting variables can be thoroughly investigated for better understanding of cell–biomaterial interactions.

Biofilm formation in total hip arthroplasty: prevention and treatment

Biomaterials science is a very active area of research, which has allowed the successful use of implants in the orthopaedic field for over a century. However, implant infection remains a clinical concern as it is associated with extensive patient morbidity and a high economic burden, which is predicted to increase due to an ageing population. Bacteria are able to adhere, colonise and develop into biofilms on the surface of biomaterials making associated infections physiologically different to other post-surgical infections. Unfortunately, biofilms exert increased protection from the host immune system and an increased resistance to antibiotic therapy in comparison to their planktonic counterparts. The aim of this review is to assess the current knowledge on treatments, pathogenesis and the prevention of infections associated with orthopaedic implants, with a focus on total hip arthroplasty.