S.C. Mendes

Bone Tissue-Engineered Implants Using Human Bone Marrow Stromal Cells: Effect of Culture Conditions and Donor Age

At present, it is well known that populations of human bone marrow stromal cells (HBMSCs) can differentiate into osteoblasts and produce bone. However, the amount of cells with osteogenic potential that is ultimately obtained will still be dependent on both patient physiological status and culture system. In addition, to use a cell therapy approach in orthopedics, large cell numbers will be required and, as a result, knowledge of the factors affecting the growth kinetics of these cells is needed. In the present study we investigated the effect of dexamethasone stimulation on the in vivo osteogenic potential of HBMSCs. After a proliferation step, the cells were seeded and cultured on porous calcium phosphate scaffolds for 1 week, and then subcutaneously implanted in nude mice for 6 weeks, in order to evaluate their in vivo bone-forming ability. Furthermore, the effect of donor age on the proliferation rate of the cultures and their ability to induce in vivo bone formation was studied. In 67% of the assayed patients (8 of 12), the presence of dexamethasone in culture was not required to obtain in vivo bone tissue formation. However, in cultures without bone-forming ability or with a low degree of osteogenesis, dexamethasone increased the bone-forming capacity of the cells. During cellular proliferation, a significant age-related decrease was observed in the growth rate of cells from donors older than 50 years as compared with younger donors. With regard to the effect of donor age on in vivo bone formation, HBMSCs from several donors in all age groups proved to possess in vivo osteogenic potential, indicating that the use of cell therapy in the repair of bone defects can be applicable irrespective of patient age. However, the increase in donor age significantly decreased the frequency of cases in which bone formation was observed.

Biocompatibility testing of novel starch-based materials with potential application in orthopaedic surgery: a preliminary study

This paper describes an extensive biocompatibility evaluation of biodegradable starch-based materials aimed at orthopaedic applications as temporary bone replacement/fixation implants. For that purpose, a polymer (starch/ethylene vinyl alcohol blend, SEVA-C) and a composite of SEVA-C reinforced with hydroxyapatite (HA) particles, were evaluated in both in vitro and in vivo assays. For the in vitro analysis cell culture methods were used. The in vivo tissue reactions were evaluated in an intramuscular and intracortical bone implantation model on goats, using light and scanning electron microscopy. A computerized image analysis system was used to obtain histomorphometric data regarding bone contact and remodelling after 6 and 12 weeks of implantation. In both in vitro and in vivo models, the SEVA-C-based materials did not induce adverse reactions, which in addition to their bone-matching mechanical properties makes them promising materials for bone replacement fixation.

Processing and in vitro Degradation of Starch/EVOH Thermoplastic Blends†

This paper describes the processing dependence of the mechanical properties of three blends of starch/ethylene vinyl alcohol (EVOH) with potential uses as biomaterials. These blends exhibit a distinct rheological behaviour and mechanical performance. Using shear controlled orientation in injection moulding (Scorim) it was possible to induce anisotropy into the mouldings and to simultaneously enhance stiffness and ductility. Degradation was studied in simulated physiological solutions (Hanks balanced salt solution) with and without added bovine serum. Both the dry weight loss and the changes in mechanical properties were determined for ageing periods up to 80 days. The degradation behaviour proved to be strongly dependent on the formulation of the material studied, and on the addition of proteins. The susceptibility of the starch/EVOH blends to degradation when sterilised with ethylene oxide (EtO) was also studied, and showed that the polymers could stand one EtO sterilisation cycle. However, two consecutive cycles severely degraded the polymer structure and properties.

Bone induction by implants coated with cultured osteogenic bone marrow cells.

The availability of osteoinductive coatings on dental and orthopedic implants will result in an improved fixation of these devices. Those cases where implants are placed in poor-quality bone or where high failure rates are obtained are especially expected to gain from such coatings. This paper presents a novel, biological approach to obtain bioactive and osteoinductive coatings on bone-replacement implant materials. This so-called tissue engineering approach utilizes osteogenic bone marrow cells that are cultured on an implant material to form a bone-like tissue. The implant materials used herein included porous calcium phosphate scaffolds and metallic plates, the latter of which were coated with a biomimetic calcium phosphate coating to facilitate cellular attachment. Bone marrow cells were obtained from a variety of species, including humans, and were grown to facilitate cellular proliferation. The cells were subsequently seeded onto the implants and cultured for an additional week to facilitate osteogenic differentiation and extracellular matrix production. The resulting hybrid implants, encompassing the biomaterial carrier and cultured bone-like tissue, were subsequently implanted subcutaneously in nude mice for 4 weeks, followed by histological examination for de novo bone formation. The results revealed that newly formed bone was seen both in porous implants and on flat metallic surfaces. This bone tissue engineering approach, therefore, offers great potential to enhance bony healing around implants in a compromised bone bed.

Evaluation of Two Biodegradable Polymeric Systems as Substrates for Bone Tissue Engineering

The aim of this study was to evaluate two biodegradable polymeric systems as scaffolds for bone tissue engineering. Rat bone marrow cells were seeded and cultured for 1 week on two biodegradable porous polymeric systems, one composed of poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) and the other composed of cornstarch blended with poly(epsilon-caprolactone) (SPCL). Porous hydroxyapatite granules were used as controls. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay and by scanning electron microscopy examination; their osteogenic differentiation was screened by the expression of alkaline phosphatase. In addition, the in vivo osteogenic potential of the engineered constructs was evaluated through ectopic implantation in a nude mouse model. Results revealed that cells were able to proliferate, differentiate, and form extracellular matrix on all materials tested. Moreover, all constructs induced abundant formation of bone and bone marrow after 4 weeks of implantation. The extent of osteogenesis (approximately 30% of void volume) was similar in all types of implants. However, the amount of bone marrow and the degree of bone contact were higher on HA scaffolds, indicating that the polymers still need to be modulated for higher osteoconductive capacity. Nevertheless, the findings suggest that both PEGT/PBT and SPCL systems are excellent candidates to be used as scaffolds for a cell therapy approach in the treatment of bone defects.

Dynamic mechanical properties of hydroxyapatite-reinforced and porous starch-based degradable biomaterials

It has been shown that blends of starch with a poly(ethylene-vinyl-alcohol) copolymer, EVOH, designated as SEVA-C, present an interesting combination of mechanical, degradation and biocompatible properties, specially when filled with hydroxyapatite (HA). Consequently, they may find a range of applications in the biomaterials field. This work evaluated the influence of HA fillers and of blowing agents (used to produce porous architectures) over the viscoelastic properties of SEVA-C polymers, as seen by dynamic mechanical analysis (DMA), in order to speculate on their performances when withstanding cyclic loading in the body. The composite materials presented a promising performance under dynamic mechanical solicitation conditions. Two relaxations were found being attributed to the starch and EVOH phases. The EVOH relaxation process may be very useful in vivo improving the implants performance under cyclic loading. DMA results also showed that it is possible to produce SEVA-C compact surface/porous core architectures with a mechanical performance similar to that of SEVA-C dense materials. This may allow for the use of these materials as bone replacements or scaffolds that must withstand loads when implanted.

In vivo bone formation by human bone marrow cells: effect of osteogenic culture supplements and cell densities

Bone marrow is known to contain a population of osteoprogenitor cells that can go through complete differentiation when cultured in a medium containing appropriate bioactive factors. In this study, porous particles of a calcium phosphate material were seeded with adult human bone marrow cells in the second passage. After an additional culture period of 1 wk in the particles, these hybrid constructs were subcutaneouslly implanted in nude mice with a survival period of 4 wk. The cell seeding densities range from 0–200 000 cells per particle and the cell culture system was designed to investigate the single and combined effects of dexamethasone and recombinant human bone morphogenetic protein 2 (rhBMP-2). The hybrid “material/tissue” constructs were processed for histology and the amount of de novo bone formation was quantified, for each culture condition, by histomorphometric techniques. The relative percentage of mineralized bone formation reached a maximal value of 19.77±5.06, for samples cultured in the presence of rhBMP-2 and with a seeding density of 200 000 cells/particle, compared to 0.52±0.45 for samples in which no cells had been cultured and had been incubated in culture medium supplemented with Dex and rhBMP-2. For the tested conditions and for the low cell numbers used in this study, rhBMP-2 proved to be an essential bioactive factor to obtain in vivo bone formation by our culture system. The results from this study prove the potential of cultured adult human bone marrow cells to initiate and accelerate de novo bone formation after transplantation into an ectopic site.

A cultured living bone equivalent enhances bone formation when compared to a cell seeding approach

The development of cell therapy methods to confer osteogenic potential to synthetic bone replacement materials has become common during the last years. At present, in the bone tissue engineering field, two different approaches use patient own cultured osteogenic cells in combination with a scaffold material to engineer autologous osteogenic grafts. One of the approaches consists of seeding cells on a suitable biomaterial, after which the construct is ready for implantation. In the other approach, the seeded cells are further cultured on the scaffold to obtain in vitro formed bone (extracellular matrix and cells), prior to implantation. In the present study, we investigated the in vivo osteogenic potential of both methods through the implantation of porous hydroxyapatite (HA) scaffolds coated with a layer of in vitro formed bone and porous HA scaffolds seeded with osteogenic cells. Results showed that as early as 2 days after implantation, de novo bone tissue was formed on scaffolds in which an in vitro bone-like tissue was cultured, while it was only detected on the cell seeded implants from 4 days onwards. In addition, after 4 days of implantation statistical analysis revealed a significantly higher amount of bone in the bone-like tissue containing scaffolds as compared to cell seeded ones.

Cultured Bone on Biomaterial Substrates

In the present work, a tissue engineering approach to treat bone defects was investigated. Such strategy was based on the use of patient own cultured bone marrow stromal cells (BMSCs) in association with biomaterials to produce autologous living bone equivalents. When engineering such implants, three main factors had to be taken into account: (i) the cells, (ii) the culture technology and (iii) the biomaterial scaffolds. The capacity of BMSCs to proliferate, differentiate along the osteogenic lineage and form a bone like tissue was demonstrated in various in vitro assays making use of biochemical, immunological, microscopic and gene expression techniques. The ability of the cells to produce bone in vivo was established using an ectopic (extra osseous) implantation model. Results indicated that BMSC cultures were composed of a heterogeneous population containing a subpopulation of cells with high proliferative capacity and with potential to differentiate into bone forming cells. Both the growth and the differentiation pattern of these cells could be manipulated, to a certain degree, through the use of bioactive factors during culture. After implantation, the bone forming capacity of the cultures proved to be related to the amount of early osteoprogenitors and precursors cells that could be induced into starting the osteogenic differentiation process. In bone marrow aspirates, this subpopulation appeared to decrease with donor age and to be strongly dependent on the donor, indicating that the aspiration procedure plays an important role in the obtained bone marrow cell population. In order to evaluate the in vivo bone formation capacity of BMSC cultures prior to implantation, an experimental method was developed in which the amount of early osteoprogenitors and precursors cells could be quantified. With regard to the technology design, data indicated that the culture of cells on the biomaterial scaffolds prior to implantation resulted in implants with faster in vivo bone forming ability as compared to scaffolds implanted shortly after cell seeding. In addition, two biodegradable polymeric systems were proposed as scaffolds to be used in the described bone engineering approach after evaluating their ability to support bone marrow cell growth, differentiation and in vivo bone formation. In summary, although the complete knowledge of the factors controlling BMSC growth and osteogenic differentiation still needs to be further expanded, the obtained results suggest that the bone tissue engineering approach described in this work presents a great potential for the repair of bone defects and will become an advantageous alternative to the traditional autologous bone grafting.