Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.

Disparate companions : tissue engineering meets cancer research

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

Host rather than graft origin of Matrigel-induced adipose tissue in the murine tissue-engineering chamber

We have recently shown that Matrigel-filled chambers containing fibroblast growth factor-2 (FGF2) and placed around an epigastric pedicle in the mouse were highly adipogenic. Contact of this construct with pre-existing tissue or a free adipose graft was required. To further investigate the mechanisms underpinning formation of new adipose tissue, we seeded these chambers with human adipose biopsies and human adipose-derived cell populations in severe combined immunodeficient mice and assessed the origin of the resultant adipose tissue after 6 weeks using species-specific probes. The tissues were negative for human-specific vimentin labeling, suggesting that the fat originates from the murine host rather than the human graft. This was supported by the strong presence of mouse-specific Cot-1 deoxyribonucleic acid labeling, and the absence of human Cot-1 labeling in the new fat. Even chambers seeded with FGF2/Matrigel containing cultured human stromal-vascular fraction (SVF) labeled strongly only for human vimentin in cells that did not have a mature adipocyte phenotype; the newly formed fat tissue was negative for human vimentin. These findings indicate that grafts placed in the chamber have an inductive function for neo-adipogenesis, rather than supplying adipocyte-precursor cells to generate the new fat tissue, and preliminary observations implicate the SVF in producing inductive factors. This surprising finding opens the door for refinement of current adipose tissue-engineering approaches.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract (Matrigel™) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 μL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Endothelial precursor cells home to a vascularized tissue engineering chamber by application of the angiogenic chemokine CXCl12

Tissue engineering of vascularized constructs has great utility in reconstructive surgery. While we have been successful in generating vascularized granulation-like tissue and adipose tissue in an in vivo tissue engineering chamber, production of other differentiated tissues in a stable construct remains a challenge. One approach is to utilize potent differentiation factors, which can influence the base tissue. Endothelial precursor cells (EPCs) have the ability to both carry differentiation factors and home to developing vasculature. In this study, proof-of-principle experiments demonstrate that such cells can be recruited from the circulation into an in vivo tissue engineering chamber. CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 was infused into the chamber through Alzet osmotic pumps and chamber cannulation between days 0 and 7, and facilitated recruitment of systemically inoculated exogenous human EPCs injected on day 6. CXCL12 infusion resulted in an eightfold increase in EPC recruitment, 2 (p = 0.03) and 7 days postinfusion (p = 0.008). Delivery of chemotactic/proliferation and/or differentiation factors and appropriately timed introduction of effective cells may allow us to better exploit the regenerative potential of the established chamber construct. © Copyright 2009, Mary Ann Liebert, Inc. 2009.

Molecular aspects of tissue engineering in the dental field

Tissue engineering is a multidisciplinary field with the potential to replace tissues lost as a result of trauma, cancer surgery, or organ dysfunction. The successful production, integration, and maintenance of any tissue-engineered product are a result of numerous molecular interactions inside and outside the cell. We consider the essential elements for successful tissue engineering to be a matrix scaffold, space, cells, and vasculature, each of which has a significant and distinct molecular underpinning (Fig. 1). Our approach capitalizes on these elements. Originally developed in the rat, our chamber model (Fig. 2) involves the placement of an arteriovenous loop (the vascular supply) in a polycarbonate chamber (protected space) with the addition of cells and an extracellular matrix such as Matrigel or endogenous fibrin (34, 153, 246, 247). This model has also been extended to the rabbit and pig (J. Dolderer, M. Findlay, W. Morrison, manuscript in preparation), and has been modified for the mouse to grow adipose tissue and islet cells (33, 114, 122) (Fig. 3)...

An endogenously deposited fibrin scaffold determines construct size in the surgically created arteriovenous loop chamber model of tissue engineering

Background: An arteriovenous loop (AVL) enclosed in a polycarbonate chamber in vivo, produces a fibrin exudate which acts as a provisional matrix for the development of a tissue engineered microcirculatory network. Objectives: By administering enoxaparin sodium - an inhibitor of fibrin polymerization, the significance of fibrin scaffold formation on AVL construct size (including the AVL, fibrin scaffold, and new tissue growth into the fibrin), growth, and vascularization were assessed and compared to controls. Methods: In Sprague Dawley rats, an AVL was created on femoral vessels and inserted into a polycarbonate chamber in the groin in 3 control groups (Series I) and 3 experimental groups (Series II). Two hours before surgery and 6 hours post-surgery, saline (Series I) or enoxaparin sodium (0.6 mg/kg, Series II) was administered intra-peritoneally. Thereafter, the rats were injected daily with saline (Series I) or enoxaparin sodium (1.5 mg/kg, Series II) until construct retrieval at 3, 10, or 21 days. The retrieved constructs underwent weight and volume measurements, and morphologic/morphometric analysis of new tissue components. Results: Enoxaparin sodium treatment resulted in the development of smaller AVL constructs at 3, 10, and 21 days. Construct weight and volume were significantly reduced at 10 days (control weight 0.337 ± 0.016 g [Mean ± SEM] vs treated 0.228 ± 0.048, [P < .001]: control volume 0.317 ± 0.015 mL vs treated 0.184 ± 0.039 mL [P < .01]) and 21 days (control weight 0.306 ± 0.053 g vs treated 0.198 ± 0.043 g [P < .01]: control volume 0.285 ± 0.047 mL vs treated 0.148 ± 0.041 mL, [P < .01]). Angiogenesis was delayed in the enoxaparin sodium-treated constructs with the absolute vascular volume significantly decreased at 10 days (control vascular volume 0.029 ± 0.03 mL vs treated 0.012 ± 0.002 mL [P < .05]). Conclusion: In this in vivo tissue engineering model, endogenous, extra-vascularly deposited fibrin volume determines construct size and vascular growth in the first 3 weeks and is, therefore, critical to full construct development.

The role of vascularization in 3-D tissue engineering

The role of vascularization in 3-D tissue engineering was studied. Mouse fat, angiogenic growth factors, adult human stem cells and fat tissue have been inserted and subsequent tissue growth was monitored. Human fat grafts or human lipoaspirates in SCID mouse chambers induced mouse fat generation at 6 weeks. Tissue engineering models utilizing intrinsic vascularization have major advantages including rapid and appropriate vascularization of new tissues.

Carbon nanostructures for hard tissue engineering

The primary goal in hard tissue engineering is to combine high-performance scaffold materials with living cells to develop biologically active substitutes that can restore tissue functions. This requires relevant knowledge in multidisciplinary fields encompassing chemical engineering, material science, chemistry, biology and nanotechnology. Here we present an overview on the recent progress of how two representative carbon nanostructures, namely, carbon nanotubes and graphene, aid and advance the research in hard tissue engineering. The article focuses on the advantages and challenges of integrating these carbon nanostructures into functional scaffolds for repairing and regenerative purposes. It includes, but is not limited to, the critical physico-chemical properties of carbon nanomaterials for enhanced cell interactions such as adhesion, morphogenesis, proliferation and differentiation; the novel designs of two- and three-dimensional nanostructured scaffolds; multifunctional hybrid materials; and the biocompatible aspects of carbon nanotubes and graphene. Perspectives on the future research directions are also given, in an attempt to shed light on the innovative and rational design of more effective biomedical devices in hard tissue engineering.

Delivery of therapeutic molecules using electrosprayed polymeric particles for applications in tissue engineering

This thesis has developed an innovative technology, electrospraying, that allows biodegradable microparticles to deliver pharmaceuticals that aid bone regeneration. The establishment, characterisation and optimisation of the technique are a step forward in developing an affordable and safe alternative to the products used currently in the clinical setting for the treatment of musculoskeletal disorders. The researcher has also investigated electrospraying as a coating technique on biodegradable structures that are used to replace damaged tissues, in order to provide localised and efficient drug delivery in the site of the defect to help tissue reconstruction.

Can bone tissue engineering contribute to therapy concepts after resection of musculoskeletal sarcoma?

Resection of musculoskeletal sarcoma can result in large bone defects where regeneration is needed in a quantity far beyond the normal potential of self-healing. In many cases, these defects exhibit a limited intrinsic regenerative potential due to an adjuvant therapeutic regimen, seroma, or infection. Therefore, reconstruction of these defects is still one of the most demanding procedures in orthopaedic surgery. The constraints of common treatment strategies have triggered a need for new therapeutic concepts to design and engineer unparalleled structural and functioning bone grafts. To satisfy the need for long-term repair and good clinical outcome, a paradigm shift is needed from methods to replace tissues with inert medical devices to more biological approaches that focus on the repair and reconstruction of tissue structure and function. It is within this context that the field of bone tissue engineering can offer solutions to be implemented into surgical therapy concepts after resection of bone and soft tissue sarcoma. In this paper we will discuss the implementation of tissue engineering concepts into the clinical field of orthopaedic oncology.

Utilizing micropellets as building blocks in cartilage tissue engineering

Osteoarthritis is the most common cause of pain and disability in Australia. This project describes a method where hundreds of cartilage microtissues are generated as tiny building blocks for assembly into larger tissues suitable for cartilage defect repair. Tissue engineering applications has the potential to overcome natural barriers and effectively repair damaged cartilage tissue. However, engineering few-millimeter thick cartilage, similar to human cartilage in the knee, remains a challenge. Utilizing micropellets as building blocks has the potential to overcome some of the challenges in cartilage tissue engineering.