Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds
Introduction
The shortcomings of autografting, with inherent donor site limitations [1], and allografting, with respect to tissue rejection [2], [3] and disease transfer [4], have inspired the development of tissue engineering in which the material properties of synthetic compounds are manipulated to enable delivery of an aggregate of dissociated cells into the host in a manner that will result in the formation of new tissue [5], [6].
The most common approach for engineering new tissues is related to cell culture and biomaterials. A desirable material for use in clinical orthopedics is a biodegradable biomimetic material that induces and promotes new bone formation by osteogenic cells at a required site. Ideally, these materials should be in the form of porous scaffolds, which provide space for tissue development and offer temporary mechanical support. Potentially suitable biomaterials for use in bone tissue engineering include ceramics (e.g. hydroxyapatite, HA, and/or tricalcium phosphate, TCP) and polymers such as poly(α-hydroxyl acids), poly(lactic acid) (PLA) [7], [8], poly(glycolic acid) [9] (PGA), or their copolymer PLGA [10], [11], [12], [13], [14]. However, the fragility of the ceramics puts them at a disadvantage as scaffold materials, while the degradation products of the synthetic polymers reduce the local pH, potentially accelerating the polymer degradation rate and inducing inflammatory response [15]. Numerous bone substitutes such as calcium metaphosphate (CMP), alginate hydrogels, polyanhydrides, polyimides, polyphosphazenes and collagen have been developed to promote bone repair and regeneration [16], [17], [18], [19], but as yet none of them are acceptable substitutes for autograft and allograft, the ‘gold standards’ in clinical practice.
The key to successful bone generation is to provide the repair site with sufficient osteogenic progenitor cells in a suitable delivery vehicle to insure osteoblastic differentiation and optimal secretory activity. Porous biomimetic matrices may provide a suitable microenvironment that promotes osteoblast proliferation and osteogenesis. To generate a biomimetic template, PLGA polymer was surface modified by signal recognition ligands to mediate the molecular and cellular response to the material, and thus promote osteoblast adhesion, spreading, growth, and differentiation [20], [21], [22]. Murphy et al. [23] reported mediation of the interior surface of porous PLGA scaffolds with a continuous bonelike mineral (BLM) layer for enhancing osteoconductivity and mechanical integrity. Du et al. [24] developed a biomimetic nano-HA/collagen (nHAC) scaffold, providing an osteogenic cells/nHAC construct characterized by good osteointegration. Other methods to improve the biomimetic qualities of biomaterials are also available.
The extracellular matrices (ECMs) of hard tissue are composed of organic and inorganic phases, the inorganic phase consisting primarily of HA crystals, and the organic phase consisting mainly of type I collagen and small amounts of ground substance including glycosaminoglycans (GAGs), proteoglycans and glycoproteins. Based on a biomimetic approach, we have prepared a composite of HA and a cross-linked network of chitosan and gelatin with glutaraldehyde [25]. HA has been proved to impart osteoconductivity to polymer scaffolds [26], and gelatin (Gel), as a partially denatured derivative of collagen, has very good biocompatibility and is biodegradable. Chitosan is a biocopolymer comprising of glucosamine and N-acetylglucosamine, obtained by deacetylation of chitin. It has been reported to be safe, hemostatic and osteoconductive, and to promote wound healing [27], [28]. Microcharacterization indicates that the presence of HA does not retard the formation of the chitosan/gelatin network, and that the polymer matrix has little influence on the high crystallinity of HA. In this paper, a highly porous three-dimensional chitosan-gelatin/hydroxyapatite (HA/CS-Gel) composite scaffold was fabricated, and the properties of rat calvarial osteoblast attachment, proliferation and differentiation were investigated.
Section snippets
Materials
HA powder, with an average particle diameter of <75 μm, was obtained from the Engineering Research Center in Biomaterials, Sichuan University (Chengdu, China). To reduce average particle diameter, an agate-ball mill was used. Both the initial and resultant compositions of the powder are pure HA [25], as shown by X-ray diffraction (XRD) and infrared (IR). The particle size and specific surface area (SSA) of HA powder were measured using a MAM500-5 powder analyzer (Ivern, UK).
Chitosan (mean
Pretreatment of the HA particles
The HA powders were pulverized to increase the SSA between the HA grains and the organic network. The HA powder obtained commercially had a wide distribution of particle sizes ranging from 0.17 to 48.27 μm diameter. After treatment by pulverization, the size distribution narrowed, and the average particle size was reduced from 19.31 to 2.80 μm (Fig. 1). For the preparation of HA/CS-Gel scaffolds, HA powders were dispersed in distilled water and ultrasonicated, resulting in an ultimate average
Discussion
Therapeutic approaches for tissue engineered repair of bone defects have attempted to mimic the natural process of bone repair by delivering a source of cells capable of differentiating into osteoblasts, inductive growth and differentiation factors, or bioresorbable scaffolding matrices to support cellular attachment, migration, and proliferation. Although logistical and regulatory issues remain to be solved, cell based therapies for the repair of clinically significant bone defects are rapidly
Conclusions
The present study demonstrated the feasibility of using the phase separation technique to fabricate biomimetic hydroxyapatite/chitosan-gelatin network (HA/CS-Gel) composites in the form of three-dimensional porous scaffolds, and further showed the adhesion, proliferation and expression of rat calvaria osteoblasts on these highly porous scaffolds. The HA/CS-Gel composite scaffolds were characterized by their biomimetic composition, and further studies on cell densities, porosities of scaffolds
Acknowledgments
The authors wish to thank the National Basic Science Research and Development Grant (973) via G1999054305 and National Science Foundation of China (Grant No. 59883002) for supporting this research.
References (34)
- et al.
Alveolar bone inductionalloplasts
Dent Clin North Am
(1980) - et al.
Bone replacement grafts. The bone substitutes
Dent Clin North Am
(1998) - et al.
Preparation and characterization of poly(l-lactic acid) foams
Polymer
(1994) - et al.
Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers
Biomaterials
(1998) - et al.
Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds
Biomaterials
(2001) - et al.
Biodegradation of PLA/GA polymersincreasing complexity
Biomaterials
(1994) - et al.
Ionically crosslinked alginate hydrogels as scaffolds for tissue engineeringPart 1. Structure, Gelation rate and mechanical properties
Biomaterials
(2001) - et al.
Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification
Bone
(2001) - et al.
An assessment of the strength of NG108-15 cell adhesion to chemically modified surfaces
Biomaterials
(1999) - et al.
Experiments on antigenicity and osteogenicity in allotransplanted cancellous bone
Int Orthop
(1990)
Allogenic transplants of bone revascularized by microvascular anastomosesa preliminary study
J Orthop Res
HIV inactivation in a bone allograft
J Periodontol
Tissue engineering
Science
Surface engineering of poly(lactic acid) by entrapment of modifying species
Macromolecules
Degradation, structure and properties of fibrous nonwoven poly(glycolic acid) scaffolds for tissue engineering
Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds
Tissue Eng
Effect of osteoblastic culture conditions on the structure of poly(dl-lactic-co-glycolic acid) foam scaffolds
Tissue Eng
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