Kristoffer Bergman

Enhanced neuronal differentiation in a three-dimensional collagen-hyaluronan matrix

Efficient 3D cell systems for neuronal induction are needed for future use in tissue regeneration. In this study, we have characterized the ability of neural stem/progenitor cells (NS/PC) to survive, proliferate, and differentiate in a collagen type I‐hyaluronan scaffold. Embryonic, postnatal, and adult NS/PC were seeded in the present 3D scaffold and cultured in medium containing epidermal growth factor and fibroblast growth factor‐2, a condition that stimulates NS/PC proliferation. Progenitor cells from the embryonic brain had the highest proliferation rate, and adult cells the lowest, indicating a difference in mitogenic responsiveness. NS/PC from postnatal stages down‐regulated nestin expression more rapidly than both embryonic and adult NS/PC, indicating a faster differentiation process. After 6 days of differentiation in the 3D scaffold, NS/PC from the postnatal brain had generated up to 70% neurons, compared with 14% in 2D. NS/PC from other ages gave rise to approximately the same proportion of neurons in 3D as in 2D (9–26% depending on the source for NS/PC). In the postnatal NS/PC cultures, the majority of βIII‐tubulin‐positive cells expressed glutamate, γ‐aminobutyric acid, and synapsin I after 11 days of differentiation, indicating differentiation to mature neurons. Here we report that postnatal NS/PC survive, proliferate, and efficiently form synapsin I‐positive neurons in a biocompatible hydrogel.

Injectable cell-free template for bone-tissue formation

Here we present a novel injectable hydrogel which forms a template for de novo formation of bone tissue. Hydrogel formation takes place in situ in less than 1 min by the cross-linking of multifunctional hyaluronic acid and polyvinyl alcohol derivatives. Endogenous cells are recruited in vivo by incorporating bone morphogenetic protein-2 (BMP-2), a powerful promoter for osteogenic differentiation. The hydrogel was evaluated in vitro by performing a cell viability test and a release study and in vivo by a rat ectopic model. Examination by X-ray, microcomputed tomography, and histology revealed a significant bone formation at the target site for gels containing BMP-2, and a complete degradation was observed for gels without BMP-2 four weeks after injection. There were no signs of inflammation or foreign body response in either group and we believe that this system has the potential as an off-the-shelf injectable to be used where bone tissue is needed.

Bone morphogenetic protein-2 delivered by hyaluronan-based hydrogel induces massive bone formation and healing of cranial defects in minipigs.

BACKGROUND Reconstruction of large craniofacial bone defects is a challenge using bone transplants or alloplastic materials. The use of bone morphogenetic protein (BMP)-2 together with a suitable carrier is an attractive option that may facilitate new bone formation. The authors have developed a hydrogel that is formed in situ by the cross-linking of multifunctional hyaluronic acid and polyvinyl alcohol derivatives mixed with hydroxyapatite nanoparticles, in the presence of BMP-2. The aim of this study was to evaluate the suitability of the hydrogel as a carrier for BMP-2 in repairing critical size cranial defects in a minipig model. METHODS Cranial defects (2 x 4 cm) were created in 14 minipigs. The experimental groups were as follows: group 1, craniotomy and application of 5 ml of hydrogel with 1.25 mg of BMP-2 (n = 6); group 2, craniotomy and application of 5 ml of hydrogel without BMP-2 (n = 6); and group 3, craniotomy with no further treatment (n = 2). RESULTS After 3 months, computed tomographic and histologic examinations were performed. There was spontaneous ossification in the untreated group, but the healing was incomplete. The hydrogel alone demonstrated no further effects. The addition of 1.25 mg of BMP-2 to the hydrogel induced a greater than 100 percent increase in bone volume (p = 0.003) and complete healing of the defects. Histologic examination revealed compact lamellar bone in the BMP group without intertrabecular fibrous tissue, as was seen in the other groups. The hydrogel was resorbed completely within 3 months and, importantly, caused no inflammatory reaction. CONCLUSION The injectable hydrogel may be favorable as a BMP-2 carrier for bone reconstruction.

Calcium phosphates compounds in conjunction with hydrogel as carrier for BMP-2 : A study on ectopic bone formation in rats

Current treatment of fractures often involves the use of bone graft or bone morphogenetic proteins (BMP) to induce fracture healing, especially in patients with a compromised healing capacity. BMP has to be delivered in conjunction with a carrier. Unfortunately, there are drawbacks and limitations with current carriers, including their bovine origin which carries the risk of an immunological response. The physical properties also limit the use to open surgical procedures, as it cannot be injected. New carriers with improved properties are therefore needed. The aim of this study was to assess the ectopic bone forming capability of various calcium phosphate compounds when used in conjunction with a hydrogel as the carrier for BMP-2. Five different ceramic additives were tested, including β-tricalcium phosphate and four types of hydroxyapatite (HAP) (nanoHAP, HAP, clods of HAP >100 μm, and the biomimetic HAP Ostim35®). The compounds were injected into the thigh muscle of rats, where it formed a gel in situ. After 4 weeks bone formation was evaluated by peripheral quantitative computed tomography and histology. The major finding was that the 20 nm nanoHAP yielded a higher bone density than the other additives (P=0.0008, ANOVA with Tukeys multiple comparison test). We hypothesize that the higher bone density induced by nanoHAP might be due to nanocrystals of calcium phosphate acting as direct building blocks for biomineralization.

In situ cross-linkable hyaluronan hydrogel enhances chondrogenesis

The present work describes the feasibility of a cross‐linkable injectable hyaluronan hydrogel for cartilage repair. The hydrogel used is a two‐component system based on aldehyde‐modified hyaluronan and hydrazide‐modified polyvinyl alcohol, which are rapidly cross‐linked in situ upon mixing. The in vitro study showed that chondrocytes and mesenchymal cells cultured in the gel form cartilage‐like tissue, rich in glycosaminoglycans, collagen type II and aggrecan. In a rabbit animal model the injection of the hydrogel improved the healing of a full‐thickness cartilage defect created in the knee as compared to non‐treated controls. This rabbit study showed that the regenerated cartilage defects stained more intensely for type II collagen upon treatment with the hydrogel. The hyaluronan‐based hydrogel may be used as a delivery vehicle for both growth factors and/or cells for cartilage repair. The in vivo study also indicated that the hydrogel alone has a beneficial effect on cartilage regeneration. Copyright

A uni-cortical femoral defect model in the rat: evaluation using injectable hyaluronan hydrogel as a carrier for bone morphogenetic protein-2.

The development of biomaterial for bone regeneration requires animal models that are reliable and designed to mimic clinically relevant situations. We have previously investigated hydrogels comprised of modified hyaluronic acid and polyvinyl alcohol in models of ectopic bone formation. This hydrogel induces bone regeneration when loaded with bone morphogenetic proteins (BMPs). To allow further optimization of hydrogels, we developed a new, femoral, non‐critical‐sized cortical defect model. In the rat femur, we drilled standardized, elongated unilateral cortical defects that did not require stabilization and that could be created bilaterally to allow paired comparisons of biomaterials. After optimizing the defect size, subsequent stress fractures occurred in only 8% and the defect healed partially over the 40 day study period. In a time‐course experiment, we treated bone defects with the previously studied hyaluronan hydrogel loaded with 10 µg hydroxyapatite and 6 µg BMP‐2. The shape of the defect allowed controlled containment of the material within the defect. The defect in the right leg was left untreated, while the left defect was filled with 40 µl of the BMP hydrogel. As determined by pQCT analysis, the treated defects had a higher bone mineral content, bone area and bone density than control defects. The relative difference was greatest between the groups at 10 and 20 days and diminished as the defect healed in the untreated legs. We conclude that this animal model allows facile and rapid screening of biomaterials for bone regeneration in cortical femoral defects without requiring external fixation. Copyright

Bone Engineering by Biomimetic Injectable Hydrogel

Osteoporosis is a multifactorial bone disease characterized by low bone mineral density (BMD) and deterioration of micro-architecture of cancellous bone leading to bone fragility and risk of fractures. In the current work, a novel tissue engineering strategy was experimented to enhance bone architecture in the risk areas via local injection of a biomimetic/osteoinductive injectable hyaluronan based hydrogel loaded with nano-hydroxyapatite crystals (Hya/HA) with/without bone morphogenetic protein (BMP-2), in distal femur of normal and ovariectomized New Zealand white rabbits. Our results revealed the osteoinductive effect of the Hya/HA composite that enhanced bone density and architecture of the rabbit distal femur.

Non-invasive tri-modal visualisation via PET/SPECT/μCT of recombinant human bone morphogenetic protein-2 retention and associated bone regeneration: A proof of concept

ABSTRACT Bone morphogenetic proteins (BMPs) are vital for bone and cartilage formation, where bone morphogenetic protein‐2 (BMP‐2) is acknowledged as a growth factor in osteoblast differentiation. However, uncontrolled delivery may result in adverse clinical effects. In this study we investigated the possibility for longitudinal and non‐invasive monitoring of implanted [125I]BMP‐2 retention and its relation to ossification at the site of implantation. A unilateral critically sized femoral defect was produced in the left limb of rats while the right femur was retained intact as a paired reference control. The defect was filled with a hyaluronan hydrogel with 25% hydroxyapatite alone (carrier control; n=2) or combined with a mixture of [125I]BMP‐2 (150&mgr;g/ml; n=4). Bone formation was monitored using micro computed tomography (&mgr;CT) scans at 1, 3, 5, 7, 9 and 12weeks. The retention of [125I]BMP‐2 was assessed with single photon emission computed tomography (SPECT), and the bone healing process was followed with sodium fluoride (Na18F) using positron emission tomography (PET) at day 3 and at week 2, 4, and 6. A rapid burst release of [125I]BMP‐2 was detected via SPECT. This was followed by a progressive increase in uptake levels of [18F]fluoride depicted by PET imaging that was confirmed as bone formation via &mgr;CT. We propose that this functional, non‐invasive imaging method allows tri‐modal visualisation of the release of BMP‐2 and the following in vivo response. We suggest that the potential of this novel technique could be considered for preclinical evaluation of novel smart materials on bone regeneration.

Preparing Bone Using an Injectable Hydrogel Scaffold

For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. In this study, we prepared crosslinked porous tubular structures from poly(trimethylene carbonate) (PTMC), in which smooth muscle cells (SMCs) were seeded and cultured in a pulsatile bioreactor mimicking the physiological conditions. PTMC was synthesized and porous tubular structures were prepared by dipping coating, cross-linking by g-irradiation, and leaching. SMCs were seeded into the porous structures by perfusion and then the constructs were cultured in a pulsatile bioreactor system. The morphologies, mechnical properties were analyzed and SMCs attachment and proliferation were evaluated by histology studies and CyQuant. Flexible tubular structures were obtained by dip coating with 3mm inner diameter and 1mm wall thickness. The porosity of the structures in wet state reached 85 vol% and the pore sizes were 60-150 mm. PTMC tubular structures showed comparable tensile strength and higher elongation compared with natural blood vessels. A pulsatile bioreactor system mimicking the conditions in vivo (dynamic pressure 70 mmHg, 75 beats/min) was successfully built. Experiements showed 7-day dilation was <10% and variation of diameter at each pulse was <1%. SMCs were homogeneously seeded in the porous scaffolds by perfusion. SMCs proliferate well to form confluent cell layer during a time period of up to 14 days, leading to constructs with even better mechanical performance. PTMC Porous tubular structures were prepared with good microstructures, elasticity and biocompatibility. SMCs were seeded and proliferated well in pulsatile bioreactor system and significant improvement of mechnical strength was observed.

Preparation and evaluation of an injectable hyaluronan hydrogel for therapeutic applications

Introduction: For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. The applicability of poly(trimethylene carbonate) (PTMC), random copolymers of TMC and e-caprolactone poly (TMC-CL), and networks based on these polymers as scaffolding materials was investigated. Methods: TMC-based (co)polymers were synthesized by ringopening polymerization. Tubular structures were prepared by dipping glass mandrels in polymer solutions containing dispersed, sieved sugar particles, followed by g-irradiation and cross-linking, and leaching. For mechanical- and biocompatibility tests, films of different thicknesses were prepared by compression molding, solvent casting, and spin-coating. Results and Discussion: PTMC and poly(TMC-CL) are flexible materials, with E-modulus values below 10 MPa and elongations at break higher than 500%. After g-irradiation in vacuo at 25– 100 kGy, networks with gel contents up to 73 wt% were obtained. The networks showed excellent creep resistance under static and dynamic loading conditions. Good cell attachment and proliferation behavior of mesenchymal stem cells, endothelial cells, and smooth muscle cells on polymer films and networks was observed. In lipase solutions, the films degraded substantially within one month by surface erosion. Porous tubular structures, with pore sizes in the range of 80 – 130 mm and a porosity of approximately 85%, could readily be prepared. A pulsatile bioreactor that allows mechanical stimulation of smooth muscle cells and endothelial cells seeded in the porous structures is being constructed. Conclusions: TMC-based (co)polymers and networks are flexible, elastic, biocompatible, and biodegradable. Porous tubular scaffolds based on these materials have much potential in tissue engineering of small diameter blood vessels.Our bodies are constantly exposed to different sorts of mechanical forces, from muscle tension to wound healing. Connective tissue adapts its extracellular matrix (ECM) to changes in mechanical load and the influence of mechanical stimulation on fibroblasts has been studied for a long time [1, 2]. When exposed to forces, fibroblasts are known to respond with expression and remodeling of ECM proteins, in particular collagen type I [3]. In this study the effect of dynamic culture conditions on human dermal fibroblasts was evaluated in terms of deposition and remodeling of ECM, with the aim of producing an ECM based scaffold. The fibroblasts were grown on compliant polymer supports either in a bioreactor with a pulsating flow or under static conditions. By applying dynamic culture conditions, the collagen deposition on the polymer supports increased fivefold. Scanning electron microscopy showed that polymer fibers were well integrated with cells and ECM and alignment along the polymer fibers was observed. Scaffold design should aim at creating structures that can help guiding the cells to form new, functional tissue. The presented system may present a new way of producing designed extracellular matrix based scaffolds for tissue engineering.Synthesis and surface activation of synthetic biodegradable polymers as support for cell produced ECMWe have previously demonstrated that porous poly-(epsiloncalprolactone) films with regularly spaced, controlled pore sizes provide adhesion and support for cultured dermal fibroblasts. We have determined the effects of applying various sized porous films (n¼3 for each treatment) on 4mm punch biopsy wounded mice to assess wounding response. Films with pores ranging in size from 3–20 microns, elicited a mild lymphocytic and foreign body perifollicular immune response, regardless of pore size but this treatment failed to significantly shorten wound healing time or increase the rate of wound closure. By 21 days after wounding the grafted porous films had become fully incorporated into or completely biodegraded in the wounded tissue. Finally, we assessed the proof of principle that live cultured fibroblasts can be delivered using porous films and sustained in model SCID mouse wounds. Human fibroblasts (30,000 cells) were subconfluently cultured on 5 micron porous films. These cell/film combinations were then transplanted onto wounded mice but failed to significantly affect wound healing. However, these transplanted fibroblast cells were readily detected using anti-human HLA antibodies in wounded SCID mice skin 21 days after treatment, when the wounds had completely healed. Taken together, these data demonstrate for the first time the feasibility of using porous films to deliver living human cells into skin wounds as part of our aim to use cell therapy to improve the wound healing response.The aim of this work is to develop an artificial artery for use in bypass surgery. The hybrid artery consists of a porous tubular scaffold made from a polyurethane elastomer. The surface of the polymer is then modified with recombinant proteins in order to encourage the growth of organised layers of vascular cells.