Functional Properties of a Purified Reconstituted Bilayer Matrix Design Support Natural Wound Healing Activities.

Biomaterial engineering has produced numerous matrices for use in tissue repair, utilizing various materials and processing methods, which can impact the ability of the products to encourage wound healing. Recently, we observed favorable clinical outcomes, using a novel purified reconstituted bilayer matrix (PRBM; Geistlich Derma-Gide) to treat chronic diabetic foot ulcers. METHODS: Evaluations of the structural and functional characteristics of PRBM in vitro were performed to assess how this biomaterial may affect the favorable clinical results observed by influencing the wound environment and key physiologic mechanisms necessary for the healing process. Investigations included scanning electron microscopy, cell culture analyses, gene expression assays, matrix metalloproteinase activity assessment, and pH measurement. RESULTS: Cross-sectional scanning electron microscopy demonstrated a distinct bilayer structure with porous and compact layers. The PRBM structure allowed cell types involved in wound healing to bind and proliferate. Expression analysis of growth factor-responsive genes demonstrated binding and preservation of bioactive growth factors TGF-ß1, bFGF, and VEGF by PRBM. Boyden chamber migration assays revealed increased cellular migration compared with controls. In the presence of PRBM, the activity of MMP-1, MMP-2, and MMP-9 was significantly lower compared with control samples. pH of the PRBM in solution was slightly acidic. CONCLUSIONS: Based on in vitro evaluations, it appears that the PRBM processing without deleterious chemical crosslinking results in a suitable ECM possessing characteristics to aid natural wound healing, including cell attachment, migration, proliferation, differentiation, and angiogenesis. These in vitro data support the promising healing rate observed clinically when chronic DFUs are treated with PRBM.

Optimising collagen scaffold architecture for enhanced periodontal ligament fibroblast migration.

Design of cell-free scaffolds for endogenous cell recruitment requires an intimate knowledge of precise relationships between structure and biological function. Here, we use morphological analysis by Micro-CT to identify the key structural features necessary for periodontal ligament fibroblast recruitment into collagen scaffolds. By the combined use of time-lapse imaging and end-point invasion analysis, we distinguish the influences of pore size, pore wall alignment, and pore transport pathways (percolation diameter) on the individual cell migration and bulk invasion characteristics of these fibroblasts. Whereas maximising percolation diameter increased individual cell speed, elongation and directionality, and produced the most rapid bulk cell invasion, a pore size of 100 µm was found to be necessary to ensure an even distribution of cells across the scaffold cross-section. These results demonstrate that control of percolation diameter and pore size may be used respectively to tune the efficiency and uniformity of invasion through macroporous scaffolds. Crucially, however, these observations were subject to the condition of pore wall alignment, with low alignment in the direction of travel producing relatively low cell speeds and limited invasion in all cases. Pore wall alignment should therefore be carefully optimised in the design of scaffolds for cell recruitment, such as that required for periodontal ligament regeneration, as a key determining factor for cell movement.

Parameterizing the Transport Pathways for Cell Invasion in Complex Scaffold Architectures.

Interconnecting pathways through porous tissue engineering scaffolds play a vital role in determining nutrient supply, cell invasion, and tissue ingrowth. However, the global use of the term "interconnectivity" often fails to describe the transport characteristics of these pathways, giving no clear indication of their potential to support tissue synthesis. This article uses new experimental data to provide a critical analysis of reported methods for the description of scaffold transport pathways, ranging from qualitative image analysis to thorough structural parameterization using X-ray Micro-Computed Tomography. In the collagen scaffolds tested in this study, it was found that the proportion of pore space perceived to be accessible dramatically changed depending on the chosen method of analysis. Measurements of % interconnectivity as defined in this manner varied as a function of direction and connection size, and also showed a dependence on measurement length scale. As an alternative, a method for transport pathway parameterization was investigated, using percolation theory to calculate the diameter of the largest sphere that can travel to infinite distance through a scaffold in a specified direction. As proof of principle, this approach was used to investigate the invasion behavior of primary fibroblasts in response to independent changes in pore wall alignment and pore space accessibility, parameterized using the percolation diameter. The result was that both properties played a distinct role in determining fibroblast invasion efficiency. This example therefore demonstrates the potential of the percolation diameter as a method of transport pathway parameterization, to provide key structural criteria for application-based scaffold design.

An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated ß-galactosidase (SA-ß-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-ß-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

Cell Invasion in Collagen Scaffold Architectures Characterized by Percolation Theory.

The relationship between biological scaffold interconnectivity and cell migration is an important but poorly understood factor in tissue regeneration. Here a scale-independent technique for characterization of collagen scaffold interconnectivity is presented, using a combination of X-ray microcomputed tomography and percolation theory. Confocal microscopy of connective tissue cells reveals this technique as highly relevant for determining the extent of cell invasion.

Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells.

INTRODUCTION: Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells. MATERIALS AND METHODS: Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry. RESULTS: MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower. CONCLUSIONS: Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.

Human MMP28 expression is unresponsive to inflammatory stimuli and does not correlate to the grade of intervertebral disc degeneration.

BACKGROUND: MMP28 (epilysin) is a recently discovered member of the MMP (matrix metalloproteinase) family that is, amongst others, expressed in osteoarthritic cartilage and intervertebral disc (IVD) tissue. In this study the hypothesis that increased expression of MMP28 correlates with higher grades of degeneration and is stimulated by the presence of proinflammatory molecules was tested. Gene expression levels of MMP28 were investigated in traumatic and degenerative human IVD tissue and correlated to the type of disease and the degree of degeneration (Thompson grade). Quantification of MMP28 gene expression in human IVD tissue or in isolated cells after stimulation with the inflammatory mediators lipopolysaccharide (LPS), interleukin (IL)-1ß, tumor necrosis factor (TNF)-α or the histondeacetylase inhibitor trichostatin A was performed by real-time RT PCR. RESULTS: While MMP28 expression was increased in individual cases with trauma or disc degeneration, there was no significant correlation between the grade of disease and MMP28 expression. Stimulation with LPS, IL-1ß, TNF-α or trichostatin A did not alter MMP28 gene expression at any investigated time point or any concentration. CONCLUSIONS: Our results demonstrate that gene expression of MMP28 in the IVD is not regulated by inflammatory mechanisms, is donor-dependent and cannot be positively or negatively linked to the grade of degeneration and only weakly to the occurrence of trauma. New hypotheses and future studies are needed to find the role of MMP28 in the intervertebral disc.