J.D. de Bruijn

Osteoinduction by calcium phosphate biomaterials

Different materials were implanted in muscles of dogs to study the osteoinduction of calcium phosphate biomaterials. Bone formation was only seen in calcium phosphate biomaterials with micropores, and could be found in hydroxyapatite (HA) ceramic, tricalcium phosphate/hydroxyapatite ceramic (BCP), β-TCP ceramic and calcium phosphate cement. The osteoinductive potential was different in different materials. The results indicate that osteoinduction can be a property of calcium phosphate biomaterials when they exhibit specific chemical and structural characteristics.

Tissue responses of calcium phosphate cement: a study in dogs.

The in vivo properties of a new kind of calcium phosphate cement were investigated in this study. Calcium phosphate cement was implanted as paste into femoral bone and dorsal muscle of dogs for 3 and 6 months, and as prehardened form into thigh muscles of dogs for 1, 2 and 6 months. Histology was performed on thin un-decalcified sections. No foreign body reaction, no inflammation and no necrosis were found both in bony site and in muscles. There was no connective tissue layer between the cement and bone when cement paste was implanted in the bone. A creeping substitution of cement by bone, in which osteoclast-like cells resorbed the cement as if the cement is a part of bone and new bone was formed directly on the resorption line of calcium phosphate cement, was found. Bone formation was found histomorphologically in pores and deep rugged surface of cement samples (both paste and prehardened form) implanted in muscles of dogs. The induced bone was also identified with backscattered scanning electron microscopy (BSE) and by energy-dispersive X-ray micro-analysis (EDX). The results suggest that the calcium phosphate cement used in this study is biocompatible, resorbable in a manner of creeping substitution, osteoconductive and osteoinductive. It seems that an ideal bone substitute can be developed by using this type of calcium phosphate cement.

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.

Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous alpha-TCP and beta-TCP.

Two kinds of tri-calcium phosphate ceramics (Ca/P = 1.50), α-TCP and β-TCP, which has the same macrostructure and microstructure, but different phase composition, were implanted in dorsal muscles of dogs. The samples were retrieved at 30, 45 and 150 days, respectively, after implantation, and were analyzed histologically. There were critically different tissue responses between α-TCP ceramic and β-TCP ceramic. Higher cell populations were observed inside the pores of β-TCP than those of α-TCP, bone tissue was found in β-TCP at 45 and 150 days, but no bone formation could be detected in any α-TCP implants in this study. On the other hand, the bone tissue in β-TCP seemed to degenerate at 150 days. The results indicate that porous β-TCP can induce bone formation in soft tissues of dogs; while the rapid dissolution of the ceramic and the higher local Ca2+, PO43- concentration due to the rapid dissolution of α-TCP may resist bone formation in α-TCP and the less rapid dissolution of β-TCP may be detrimental to already formed bone in β-TCP.

Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

A comparison of the osteoinductive potential of two calcium phosphate ceramics implanted intramuscularly in goats.

The osteoinductive potential, or bone induction potency, of two calcium phosphate ceramics was evaluated after intramuscular implantation in goats. The ceramics were comprised of hydroxyapatite (HA) and biphasic calcium phosphate (BCP), the later of which contained a 85/15 mixture of hydroxyapatite and tricalcium phosphate (TCP). Both ceramics had a similar macroporosity of around 55% and a pore distribution between 100 and 800 μm. Besides the difference in chemistry, BCP was also microporous and hence had a different surface microstructure. After implantation in the back muscles of four goats for 12 weeks, all 8 BCP samples (7×7×7 mm3) showed the presence of bone formation in the macropores (1±1%), while no bone was found in any of the HA samples. The used BCP can therefore be characterized as an osteoinductive material. Having the ability to induce bone formation in soft tissues, the BCP presented herein may be a useful biomaterial for bone repair when combined with cultured osteogenic cells, growth factors or both.

Cytocompatibility and response of osteoblastic-like cells to starch-based polymers: effect of several additives and processing conditions

This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds. It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications. This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above. The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines. A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface. In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterials.

Structural arrangements at the interface between plasma sprayed calcium phosphates and bone

Plasma sprayed coatings of tetracalcium phosphate, magnesium whitlockite and three types of hydroxyapatite, varying in degree of crystallinity, were evaluated with light microscopy, scanning electron microscopy and backscatter electron microscopy (BSE) after implantation periods of 1, 2 and 4 wk in rat femora. BSE revealed that both tetracalcium phosphate and semi-crystalline hydroxyapatite underwent distinct bulk degradation and loss of relatively large particles. Amorphous hydroxyapatite showed a gradual surface degradation, indicated by a transition zone varying in grey level between that of the coating and bone tissue, while degradation was negligible with the highly crystalline material and magnesium whitlockite. Degradation appeared to be related to bone apposition, since more bone seemed to be present on amorphous hydroxyapatite and tetracalcium phosphate, as compared to highly crystalline hydroxyapatite and magnesium whitlockite coatings. At the interface between bone and magnesium whitlockite, a seam of unmineralized bone-like tissue was frequently seen with light microscopy, while few areas with bone contact were present. X-ray microanalysis revealed that both the magnesium whitlockite coating and the unmineralized bone-like tissue contained substantial amounts of aluminium which, in addition to possible influences of magnesium, may have caused the impaired mineralization. The results of this preliminary study indicate that, with regard to early bone formation, amorphous hydroxyapatite coatings seem to be beneficial over highly crystalline coatings. However, further experiments should be performed to give conclusive data on (i) the statistical significance of the differences in bone apposition rate, and (ii) the long-term behaviour of both amorphous and highly crystalline coatings in bone and their relation to implant performance.

Expansion of mesenchymal stem cells using a microcarrier‐based cultivation system: growth and metabolism

For the continuous and fast expansion of mesenchymal stem cells (MSCs), microcarriers have gained increasing interest. The aim of this study was to evaluate the growth and metabolism profiles of MSCs, expanded in a microcarrier‐based cultivation system. We investigated various cultivation conditions to expand goat mesenchymal stem cells on Cytodex 1 microcarriers. These conditions differed in feeding regime, i.e. the addition of fresh proliferation medium, with or without new microcarriers. For all conditions, cell attachment, cell proliferation, energy source consumption, metabolite production, and cell distribution on the microcarriers were studied. Attachment efficiencies of 40% were obtained followed by successful expansion up to 15 cultivation days. Depending on the feeding regime, an exponential growth, stationary growth, and decline growth phase could be distinguished. Addition of 30% fresh medium containing microcarriers every three days showed the longest continuous proliferation of goat MSCs on microcarriers. This feeding regime has the advantage that metabolites, such as ammonia, are diluted and that new energy sources, such as glucose and glutamine, and additional surface area are provided to the cells. In addition, by adding extra microcarriers a more homogenous cell distribution on the microcarriers is obtained as a result of bead‐to‐bead transfer. A correlation between nutrient consumption, metabolite production and cell growth was observed. The decreasing yield of lactate from glucose over time indicated a possible shift in cellular metabolism. Copyright

Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism

Adult stem cells, or mesenchymal stromal cells (MSCs), are of great potential for cell therapy and tissue‐engineering applications. However, for therapeutic use, these cells need to be isolated from tissue or a biopsy and efficiently expanded, as they cannot be harvested in sufficient quantities from the body. In our opinion, efficient expansion of MSCs can be achieved in a microcarrier‐based cultivation system. This study selected a suitable microcarrier for human bone marrow‐derived stromal cells (HBMSCs), optimized cell‐seeding strategies by varying serum concentrations, and optimized dynamic expansion of the HBMSCs in a microcarrier‐based spinner flask cultivation system by applying various feeding regimes. Cytodex 1 microcarriers in combination with a low‐serum concentration (0–5%) in the medium resulted in the highest seeding efficiency for the HBMSCs. Subsequently, significant expansion of the HBMSCs on these carriers has been observed. The highest number of HBMSCs population doublings (4.8 doublings) was obtained by a combination of 50% medium refreshment combined with addition of 30% medium containing microcarriers every 3 days. Exponential cell growth was observed for at least 9 days after seeding, provided that sufficient nutrients (such as glucose) were present, metabolite concentrations (such as ammonia) were kept below growth‐inhibitory concentrations and adequate surface area was present for the cells. After dynamic expansion of the HBMSCs, the cells retained their differentiation potential and their cell surface markers, indicating that HBMSCs expansion on Cytodex 1 microcarriers did not alter the phenotypic properties of the cells. Copyright