Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds

Abstract

A novel biodegradable hydroxyapatite/chitosan-gelatin network (HA/CS-Gel) composite of similar composition to that of normal human bone was prepared as a three-dimensional biomimetic scaffold by phase separation method for bone tissue engineering. Changing the solid content and the compositional variables of the original mixtures allowed control of the porosities and densities of the scaffolds. The HA granules were dispersed uniformly in the organic network with intimate interface contact via pulverizing and ultrasonically treating commercial available HA particles. Scaffolds of 90.6% porosity were used to examine the proliferation and functions of the cells in this three-dimensional microenvironment by culturing neonatal rat caldaria osteoblasts. Histological and immunohistochemical staining and scanning electron microscopy observation indicated that the osteoblasts attached to and proliferated on the scaffolds. Extracellular matrices including collagen I and proteoglycan-like substrate were synthesized, while osteoid and bone-like tissue formed during the culture period. Furthermore, the cell/scaffold constructs had good biomineralization effect after 3 weeks in culture.

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.