Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering

Osteoblast adhesion on biomaterials.

Control of stem cell fate by physical interactions with the extracellular matrix.

Although several major progresses have been introduced in the field of bone regenerative medicine during the years, current therapies, such as bone grafts, still have many limitations. Moreover, and in spite of the fact that material science technology has resulted in clear improvements in the field of bone substitution medicine, no adequate bone substitute has been developed and hence large bone defects/injuries still represent a major challenge for orthopaedic and reconstructive surgeons. It is in this context that TE has been emerging as a valid approach to the current therapies for bone regeneration/substitution. In contrast to classic biomaterial approach, TE is based on the understanding of tissue formation and regeneration, and aims to induce new functional tissues, rather than just to implant new spare parts. The present review pretends to give an exhaustive overview on all components needed for making bone tissue engineering a successful therapy. It begins by giving the reader a brief background on bone biology, followed by an exhaustive description of all the relevant components on bone TE, going from materials to scaffolds and from cells to tissue engineering strategies, that will lead to "engineered" bone. Scaffolds processed by using a methodology based on extrusion with blowing agents.

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Bone Tissue Engineering: State of the Art and Future Trends

http://www.wissenschaftsdialog.de/34Caplan%20MSC2001.pdf

Mesenchymal stem cells: building blocks for molecular medicine in the 21st century

We employed culture technology, which provides bone tissue in vitro, to expand and promote the osteogenic ability of marrow cells in porous hydroxyapatite (HA). Marrow cells were obtained from rat femur and cultured in a standard medium for 10 days, then trypsinized to make composites of HA and the cells. An additional 2-week culture (subculture) was done for the composite in a standard medium with or without the addition of dexamethasone (Dex). The 2-week subcultured composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested and prepared for the biochemical analysis of alkaline phosphatase activity and bone Gla protein content, as well as histological analysis of decalcified and undecalcified sections. In Dex-treated composites, high alkaline phosphatase activity could be detected 1 week after implantation and was maintained until 8 weeks after implantation. The bone Gla protein content could also be detected 1 week after implantation, followed by a steady increase with the passage of time until 8 weeks after implantation. The histological analysis showed active bone formation even 1 week after implantation. The bone formation was evidenced by active osteoblast lining and the appearance of calcein labeling following calcein injection 1 week after implantation. Thus, Dex-treated subcultured marrow cells in pore regions of HA showed a high osteogenic response immediately after transplantation. In contrast, Dex-untreated composite did not show bone formation and contained traces of these biochemical parameters. These results indicate that the inherent osteogenic ability of marrow stromal stem cells in pore regions of HA can be stimulated using tissue culture technology; and thus, formed osteogenic HA can show immediate osteoblastic activity in in vivo situations, suggesting the applicability of the HA in clinical situations.

Immediate bone forming capability of prefabricated osteogenic hydroxyapatite

This study determined the bone formation in porous calcium carbonate (CC) and porous hydroxyapatite (HA) in ectopic sites. The bone formation stimulus was derived from bone marrow cells. CC and HA in the shape of disks were implanted with or without rat marrow cells into subcutaneous sites of syngeneic rats. The CC and HA had identical microstructure: pore size was 190-230 microns, porosity was 50-60% and they were fully interconnected. Bone did not form in any implants without marrow cells (disks themselves), whereas bone consistently formed in the pores of all implants with marrow cells after 4 weeks. The bone formation of both CC and HA occurred initially on surface of the pore regions and progressed toward the center of the pore. Scanning electron microscopy and electron-probe microanalysis revealed a continuum of calcium at the interfaces of both bone/CC and bone/HA implants. These results indicate that the bone formation in calcium carbonate derived from marine corals is comparable to the bioactive hydroxyapatite.

Bone formation process in porous calcium carbonate and hydroxyapatite.

To investigate the significance of apatite-wollastonite-containing glass ceramic (AW ceramic) surfaces and the biological apatite layer formed on these surfaces, rat marrow cell culture, which shows osteogenic differentiation, was carried out on four different culture substrata (control culture dish, two AW ceramics, each having a different surface roughness, and a ceramic on which an apatite layer was formed. A culture period of 2 weeks in the presence of β-glycerophosphate, ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain on all substrata. The stain on the apatite-formed AW ceramic was the most intense, the enzyme activity being about twice that of the control culture dish, which had the lowest stain and activity of the four substrata. Northern blot analysis of bone Gla protein (BGP) showed the same tendency, that is, the amount of BGP mRNA from cultured cells on the apatite-formed AW ceramics was the highest and the mRNA on the control dish was the lowest. These data indicate that the glass ceramic surface promotes osteoblastic differentiation and that the promotion can be further enhanced by the formation of a biological apatite layer on the ceramic surface. © 1996 John Wiley & Sons, Inc.

Osteogenic differentiation of cultured marrow stromal stem cells on the surface of bioactive glass ceramics

The effect of aging on osteoblastic differentiation of marrow stromal stem cells was examined. Porous hydroxyapatite (HA) disks were soaked in cells suspensions of bone marrow cells from young (8 weeks) and old rats (60 weeks) and then implanted subcutaneously in syngeneic young and old rats. The bone marrow/HA composites were harvested 8 weeks later, and the contents of bone Gla protein (BGP) and alkaline phosphatase (ALP) activity in them were determined. Histologically, bone formation could be detected in all the composites in young recipient rats; however, some old bone marrow/HA composites in old recipients did not show bone formation and the bone volume in the young bone marrow/HA composites was greater than in the old bone marrow/HA composites. The ratios of ALP activities of young bone marrow/HA composites to old bone marrow/HA composites in young and old recipients were about five times and four times, respectively. The ratios of BGP contents of young bone marrow/HA to old bone marrow/HA composite in young and old recipients were about nine and eight times, respectively. The results suggest that the decreased bone formation observed in old bone marrow cells was due to a smaller population of stromal cells and/or decreased capacity of differentiation of stromal stem cells into osteogenic cells.

https://asbmr.onlinelibrary.wiley.com/doi/pdf/10.1359/jbmr.1997.12.6.989

The Effect of Aging on Bone Formation in Porous Hydroxyapatite: Biochemical and Histological Analysis

BACKGROUND Bone marrow cells differentiate into bone-forming osteoblasts when cultured in medium supplemented with 15% fetal bovine serum, ascorbic acid, beta-glycerophosphate, and dexamethasone. METHODS To investigate in vivo osteoblastic activity and bone matrix formation by cultured bone marrow cells, Fischer rat marrow cells were cultured for 2 weeks in porous hydroxyapatite (HA) and then subcutaneously implanted into 7-week-old male syngeneic rats. The implants were harvested after 8 and 52 weeks for biochemical and histological analyses. RESULTS At both times, formation of lamellar bone accompanied by regeneration of marrow were seen in many of the HA pores. When a fluorochrome (calcein) was administered at 50 weeks after implantation, it was detected in the pores of implants harvested at 52 weeks. Osteoclastic resorption followed by new bone formation was seen in some pores at 52 weeks, indicating that bone remodeling was continuing. The alkaline phosphatase activity of implants harvested at 52 weeks was comparable to that at 8 weeks, whereas the osteocalcin content of the implants harvested at 52 weeks was about twice that at 8 weeks. CONCLUSION These results demonstrated that there was persistent in vivo osteogenic and hematopoietic activity in the prefabricated bone/HA constructs, and indicated that normal bone tissue was regenerated after grafting of the constructs, which were brittle before implantation. Tissue engineering using HA and cultured marrow cells culture may provide an alternative method of bone transplantation for patients with skeletal disorders, although further in vivo and in vitro experiments are needed.

Takafumi Yoshikawa

Papers

Immediate bone forming capability of prefabricated osteogenic hydroxyapatite

Bone formation process in porous calcium carbonate and hydroxyapatite.

Osteogenic differentiation of cultured marrow stromal stem cells on the surface of bioactive glass ceramics

The Effect of Aging on Bone Formation in Porous Hydroxyapatite: Biochemical and Histological Analysis

In vivo osteogenic durability of cultured bone in porous ceramics: a novel method for autogenous bone graft substitution.