Henrik Bäckdahl

Microporous bacterial cellulose as a potential scaffold for bone regeneration

Nanoporous cellulose biosynthesized by bacteria is an attractive biomaterial scaffold for tissue engineering due to its biocompatibility and good mechanical properties. However, for bone applications a microscopic pore structure is needed to facilitate osteoblast ingrowth and formation of a mineralized tissue. Therefore, in this study microporous bacterial cellulose (BC) scaffolds were prepared by incorporating 300-500 microm paraffin wax microspheres into the fermentation process. The paraffin wax microspheres were subsequently removed, and scanning electron microscopy confirmed a microporous surface of the scaffolds while Fourier transform infrared spectroscopy verified the elimination of paraffin and tensile measurements showed a Youngs modulus of approximately 1.6 MPa. Microporous BC and nanoporous (control) BC scaffolds were seeded with MC3T3-E1 osteoprogenitor cells, and examined by confocal microscopy and histology for cell distribution and mineral deposition. Cells clustered within the pores of microporous BC, and formed denser mineral deposits than cells grown on control BC surfaces. This work shows that microporous BC is a promising biomaterial for bone tissue engineering applications.

Engineering microporosity in bacterial cellulose scaffolds.

The scaffold is an essential component in tissue engineering. A novel method to prepare three‐dimensional (3D) nanofibril network scaffolds with controlled microporosity has been developed. By placing paraffin wax and starch particles of various sizes in a growing culture of Acetobacter xylinum, bacterial cellulose scaffolds of different morphologies and interconnectivity were prepared. Paraffin particles were incorporated throughout the scaffold, while starch particles were found only in the outermost area of the resulting scaffold. The porogens were successfully removed after culture with bacteria and no residues were detected with electron spectroscopy for chemical analysis (ESCA) or Fourier transform infra‐red spectroscopy (FT–IR). Resulting scaffolds were seeded with smooth muscle cells (SMCs) and investigated using histology and organ bath techniques. SMC were selected as the cell type since the main purpose of the resulting scaffolds is for tissue engineered blood vessels. SMCs attached to and proliferated on and partly into the scaffolds. Copyright

Behavior of human chondrocytes in engineered porous bacterial cellulose scaffolds

Regeneration of articular cartilage damage is an area of great interest due to the limited ability of cartilage to self-repair. The latest cartilage repair strategies are dependent on access to biomaterials to which chondrocytes can attach and in which they can migrate and proliferate, producing their own extracellular matrix. In the present study, engineered porous bacterial cellulose (BC) scaffolds were prepared by fermentation of Acetobacter xylinum (A. xylinum) in the presence of slightly fused wax particles with a diameter of 150-300 microm, which were then removed by extrusion. This porous material was evaluated as a scaffold for cartilage regeneration. Articular chondrocytes from young adult patients as well as neonatal articular chondrocytes were seeded with various seeding techniques onto the porous BC scaffolds. Scanning electron microscopy (SEM) analysis and confocal microscopy analysis showed that cells entered the pores of the scaffolds and that they increasingly filled out the pores over time. Furthermore, DNA analysis implied that the chondrocytes proliferated within the porous BC. Alcian blue van Gieson staining revealed glycosaminoglycan (GAG) production by chondrocytes in areas where cells were clustered together. With some further development, this novel biomaterial can be a suitable candidate for cartilage regeneration applications.

Small calibre biosynthetic bacterial cellulose blood vessels: 13-months patency in a sheep model

Abstract Objectives. Many patients in need of bypass surgery lack graft material and current synthetic alternatives have poor performance. A 4 mm vascular graft composed of bacterial cellulose (BC) was developed and tested in pilot study in a large animal model. Design. BC is a biopolymer made by the bacteria acetobacter xylinum. BC grafts (n = 16) with 4 cm length and 4 mm internal diameter were implanted bilaterally in the carotid arteries of eight sheep. No long-term antithrombotic therapy was administered. Patency was assessed with ultrasound. Histology, immunohistochemistry, and electron microscopy were performed after explantation. Results. Fifty percent of the grafts occluded within two weeks. One animal died with patent grafts after 14 days. In the three remaining animals 5/6 grafts were patent after nine months. Two animals were followed 13 months after implantation with 3/4 grafts patent at explantation. All patent grafts had confluent endothelial-like cells. Conclusions. Biosynthetic small calibre vascular grafts made from BC can be patent for up to 13 months in sheep carotid arteries. BC is a potential material for small calibre grafts but patency in animal models needs to be improved before clinical studies can be planned.

Recellularization of Acellular Human Small Intestine Using Bone Marrow Stem Cells

We aimed to produce an acellular human tissue scaffold with a view to test the possibility of recellularization with bone marrow stem cells to produce a tissue‐engineered small intestine (TESI). Human small‐bowel specimens (n = 5) were obtained from cadaveric organ donors and treated sequentially with 6% dimethyl sulfoxide in hypotonic buffer, 1% Triton X‐100, and DNase. Each small intestine (SI) piece (6 cm) was recellularized with EPCAM+ and CD133+ allogeneic bone marrow stem cells. Histological and molecular analysis demonstrated that after decellularization, all cellular components and nuclear material were removed. Our analysis also showed that the decellularized human SI tissue retained its histoarchitecture with intact villi and major structural proteins. Protein films of common extracellular matrix constituents (collagen I, laminin, and fibronectin) were found in abundance. Furthermore, several residual angiogenic factors were found in the decellularized SI. Following recellularization, we found viable mucin‐positive goblet cells, CK18+ epithelial cells in villi adjacent to a muscularis mucosa with α‐actin+ smooth muscle cells, and a high repopulation of blood vessels with CD31+ endothelial cells. Our results show that in the future, such a TESI would be ideal for clinical purposes, because it can be derived from the recipients own immunocompatible bone marrow cells, thus avoiding the use of immunosuppression.

Towards the development of a bioengineered uterus: Comparison of different protocols for rat uterus decellularization

Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H2O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH2O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.

In Vivo Application of Tissue-Engineered Veins Using Autologous Peripheral Whole Blood: A Proof of Concept Study.

Vascular diseases are increasing health problems affecting > 25 million individuals in westernized societies. Such patients could benefit from transplantation of tissue-engineered vascular grafts using autologous cells. One challenge that has limited this development is the need for cell isolation, and risks associated with ex vivo expanded stem cells. Here we demonstrate a novel approach to generate transplantable vascular grafts using decellularized allogeneic vascular scaffolds, repopulated with peripheral whole blood (PWB) in vitro in a bioreactor. Circulating, VEGFR-2 +/CD45 + and a smaller fraction of VEGFR-2 +/CD14 + cells contributed to repopulation of the graft. SEM micrographs showed flat cells on the luminal surface of the grafts consistent with endothelial cells. For clinical validation, two autologous PWB tissue-engineered vein conduits were prepared and successfully used for by-pass procedures in two pediatric patients. These results provide a proof of principle for the generation of transplantable vascular grafts using a simple autologous blood sample, making it clinically feasible globally.

An Alternative Approach to Decellularize Whole Porcine Heart

Abstract Scaffold characteristics are decisive for repopulating the acellular tissue with cells. A method to produce such a scaffold from intact organ requires a customized decellularization protocol. Here, we have decellularized whole, intact porcine hearts by serial perfusion and agitation of hypotonic solution, an ionic detergent (4% sodium deoxycholate), and a nonionic detergent (1% Triton X-100). The resultant matrix was characterized for its degree of decellularization, morphological and functional integrity. The protocol used resulted in extensive decellularization of the cardiac tissue, but the cytoskeletal elements (contractile apparatus) of cardiomyocytes remained largely unaffected by the procedure although their membranous organelles were completely absent. Further, several residual angiogenic growth factors were found to be present in the decellularized tissue.

Successful tissue engineering of competent allogeneic venous valves

OBJECTIVE The purpose of this study was to evaluate whether tissue-engineered human allogeneic vein valves have a normal closure time (competency) and tolerate reflux pressure in vitro. METHODS Fifteen human allogeneic femoral vein segments containing valves were harvested from cadavers. Valve closure time and resistance to reflux pressure (100 mm Hg) were assessed in an in vitro model to verify competency of the vein valves. The segments were tissue engineered using the technology of decellularization (DC) and recellularization (RC). The decellularized and recellularized vein segments were characterized biochemically, immunohistochemically, and biomechanically. RESULTS Four of 15 veins with valves were found to be incompetent immediately after harvest. In total, 2 of 4 segments with incompetent valves and 10 of 11 segments with competent valves were further decellularized using detergents and DNAse. DC resulted in significant decrease in host DNA compared with controls. DC scaffolds, however, retained major extracellular matrix proteins and mechanical integrity. RC resulted in successful repopulation of the lumen and valves of the scaffold with endothelial and smooth muscle cells. Valve mechanical parameters were similar to the native tissue even after DC. Eight of 10 veins with competent valves remained competent even after DC and RC, whereas the two incompetent valves remained incompetent even after DC and RC. The valve closure time to reflux pressure of the tissue-engineered veins was <0.5 second. CONCLUSIONS Tissue-engineered veins with valves provide a valid template for future preclinical studies and eventual clinical applications. This technique may enable replacement of diseased incompetent or damaged deep veins to treat axial reflux and thus reduce ambulatory venous hypertension.