Development of collagen condensation method to improve mechanical strength of tissue engineering scaffolds

M A TE RI A L S CH A RACT ER IZ A TI O N 61 ( 20 1 0 ) 9 0 7 –9 1 1

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Development of collagen condensation method to improve mechanical strength of tissue engineering scaffolds Shunji Yunoki a,⁎, Toshiyuki Ikomab , Junzo Tanakab a b

Life Science Group, Tokyo Metropolitan Industrial Technology Research Institute, 2-11-1 Fukasawa, Setagaya-ku, Tokyo 158-0081, Japan Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, 2-12-1-S7-1, Ookayama, Meguro-ku, Tokyo 152-8550, Japan

AR TIC LE D ATA

ABSTR ACT

Article history:

A major drawback of collagen sponges regarding their use in tissue engineering scaffolds is

Received 8 December 2009

their weak mechanical properties under wet conditions. To overcome this problem without

Received in revised form 18 May 2010

the use of other skeletal materials, the exhaustive condensation technique of reconstituted

Accepted 18 May 2010

collagen fibrils was developed to fabricate high-density collagen sponges using freeze drying. The density linearly increased with an increase in the concentration of collagen

Keywords:

fibrils. The compression tests under wet conditions demonstrated that the toughness and

Collagen

stiffness of the collagen sponges increased with an increase in the density. The collagen

Tissue engineering

sponge with a density of 129 mg/cm3 showed a compressive strength (to a strain of 30%) of

Scaffold

8.88 kPa and a modulus of 332 kPa. These results suggested the possibility that the

Sponge

mechanical properties of collagen sponges can increase significantly while retaining the

Mechanical property

sponges' inherent bioresorbability. © 2010 Elsevier Inc. All rights reserved.

1.

Introduction

Tissue engineering is a field of research that aims to replace organs [1] and requires a suitable scaffold for doing so. Several three-dimensional materials with different pore structures have been developed as potential scaffolds [2]. Among these materials, type I collagen (abbreviated here as “collagen”) with spongy forms has been used widely for this purpose because of its many advantages including bioresorbability and biocompatibility [3]. Collagen sponges have frequently been combined with biopolymers and bioceramics according to their applications; however, the weak mechanical properties of collagen sponges present serious disadvantages for practical application [4], especially under a wet condition [5]. Besides the collagen sponges, porous synthetic polymers such as polyglycolic acid (PGA) and poly(DL-lactic-co-glycolic acid) (PLGA) have been studied extensively as scaffolds [6,7] in

spite of their relatively low biodegradability and cell affinities. Their main benefit is their superior mechanical strength due to their hydrophobic properties. Hybrids of collagen and synthetic polymers for use as skeletal materials have also been developed [5,8–10]. However, their biodegradability is too low to achieve complete regeneration of damaged tissues within several months [9]. We believe that the collagen sponge without synthetic polymers is an ideal scaffold for dermal, bone, and cartilage tissue engineering. Its use is undoubtedly limited, however, by its weak mechanical properties. The collagen sponges with low mechanical properties have been fabricated from collagen solutions at low concentrations below 1% [11,12] and are one of the major scaffolds. The increase in density of the collagen sponge is expected to improve mechanical properties; however, fabrication methods of highdensity collagen sponges have not yet been investigated. In the present study, we investigated the exhaustive condensation of

⁎ Corresponding author. Tel.: +81 3 3702 3115; fax: + 81 3 3703 9768. E-mail address: [email protected] (S. Yunoki). 1044-5803/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.matchar.2010.05.010

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reconstituted collagen fibrils to improve mechanical properties of collagen sponges. In particular the porous structure and the mechanical properties concerning the compressive strength and modulus were analyzed.

2.

Experimental

To prepare a condensed monomeric collagen suspension, a solution of collagen from porcine dermis (Nitta Gelatin) was dialyzed against de-ionized water and freeze dried. De-ionized water was added to the dried spongy collagen to achieve concentrations of 3 and 5%, thus providing transparent and highly viscous suspensions. To reconstitute the collagen fibrils, the monomeric collagen solution was mixed with neutral phosphate buffer and incubated overnight at 37 °C to form a gel of collagen fibrils. The gel was centrifuged at 10,000 ×g for 20 min. The precipitated collagen fibrils were collected and homogenized using a homogenizer to promote dehydration, followed by additional centrifugation. This cycle was repeated three times, resulting in a condensed collagen fibril suspension. The concentration of the suspension was determined based on the dry content of collagen (n = 3). The suspension thus obtained was diluted with de-ionized water and re-homogenized to achieve concentrations of 4, 6, 8, and 10%. The collagen sponges (14 mm diameter, 9 mm height) were prepared from both the collagen fibril and monomeric collagen suspensions by freeze drying as described in our previous method [13]. The density of the collagen sponges obtained was determined from the weight and volume of the cylindrical samples (n = 4). The sponges prepared from the collagen fibril suspensions were cross-linked by two methods according to our previous report [12]: 1) dehydrothermal treatment at 130 °C for 24 h in vacuo (DHT treatment); or 2) the DHT treatment followed by swelling in 50 mM 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) solution in ethanol (EDC treatment). Morphological analyses of the crosssectional porous structures of the collagen sponges that underwent the DHT treatment were performed using JSM6360LA (JEOL) scanning electron microscopy (SEM) at an accelerated voltage of 15 kV. Compression tests for the collagen sponges were performed by a mechanical tester. The cylindrical collagen sponges were soaked in phosphate buffer saline (PBS), and stored at 4 °C for 24 h. The specimens (n = 5) were compressed using a cylindrical probe (20 mm diameter) at a cross-head speed of 10 mm/min to achieve a strain of 50% and immediately returned to the initial position. The cyclic tests were repeated 10 times at intervals of 60 s. Mechanical data were determined from the stress–strain (S–S) curve of the third cycle. Compressive modulus was calculated from the slope of the stress–strain curve in the linear region (strain from 0.04 to 0.08).

3.

Results

3.1.

Preparation of Collagen Suspensions and Sponges

The concentration of the condensed collagen fibril suspensions reached 11.6% through repeated homogenization and

centrifugation, which enabled us to prepare suspensions with various concentrations (4, 6, 8, and 10%) by the addition of water. The suspensions were highly viscous, but could be put into freezing containers while minimizing internal air bubbles. On the other hand, it was difficult to remove air bubbles from the monomeric collagen suspensions at concentrations above 5% because of their extreme stickiness. Thus, the suspensions at the concentrations of 3% and 5% were used to prepare the collagen sponges. Fig. 1 shows the densities of the collagen sponges prepared by freeze drying. The density linearly increased with an increase in the concentrations of the suspensions (SD < 2 mg/ cm3). The correlation appeared to be similar in the collagen sponges prepared from each type of collagen suspension.

3.2.

Porous Structures

Fig. 2 shows the cross-sectional SEM images of the collagen sponges prepared from condensed collagen fibril suspensions with the DHT treatment. The cross-sectional morphology showed porous structures. Although SEM observations are quick analyses and do not provide quantitative data for pore sizes, it can be seen that the pore sizes ranged widely from about 100 µm to several hundred µm. The interconnectivity between the pores tended to be decreased by increasing the concentration of the suspension. The pore structures of the collagen sponges that received the EDC treatment were similar to those receiving the DHT treatment as reported previously (data not shown) [13].

3.3.

Mechanical Properties

The mechanical properties of the collagen sponges were evaluated by repeated cyclical compression tests performed in PBS. The 10 repetitions of compressions to a strain of 50% gave almost the same S–S curves, in which only the first cycle showed slightly higher stresses. No breaking points were observed. Fig. 3a shows the compressive strength of the collagen sponges. The strength linearly increased with increases in the

Fig. 1 – Density of collagen sponges prepared from monomeric collagen suspensions (open circles) and collagen fibril suspensions (closed circles) in various concentrations. Data are given as mean (n = 4). SD of all data was below 2 mg/cm3.

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Fig. 2 – Cross-sectional SEM images of the collagen sponges prepared from the condensed collagen fibril suspensions of which the concentrations are (a) 4%, (b) 6%, (c) 8%, and (d) 10%. Figures (e), (f), (g), and (h) are magnifications of (a), (b), (c), and (d), respectively. Bars in the figures indicate 100 μm.

density. The collagen sponges that underwent the DHT and the EDC treatments showed compressive strengths of 4.31 kPa and 8.88 kPa in densities of 136 mg/cm3 and 129 mg/cm3, respectively. Corresponding results were obtained for the compressive modulus (Fig. 3b), showing compressive moduli of 154 kPa and 332 kPa in densities of 136 mg/cm3 and 129 mg/ cm3, respectively.

4.

Discussion

The use of collagen for porous sponges is currently experiencing a renaissance for tissue engineering techniques including dermal, bone, and cartilage repairs. The collagenbased sponges combined with biopolymers or bioceramics

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Fig. 3 – Compressive strength (a) and modulus (b) of the collagen sponges prepared from the collagen fibril suspensions as a function of density. Closed squares: DHT treatment; open squares: EDC treatment. Data are given as mean ± SD (n = 5).

have also been investigated. Especially in bone tissue engineering, the composites of osteoconductive hydroxyapatite (HAp) and collagen have been extensively studied as promising scaffolding materials [14,15], in which HAp plays a crucial role on bone regeneration and mechanical properties. Collagen sponges with an osteoinductive human bone morphogenetic protein demonstrate bone regeneration without the use of osteoconductive bioceramics [16]. For both pure collagen and collagen-based composite sponges, the main drawback in their practical use is their weakness of mechanical properties. The collagen fibril suspension was exhaustively condensed by three-times-repeated homogenization and centrifugation, which enabled us to prepare high-density collagen sponges with high mechanical properties (toughness and stiffness). As for previous collagen sponges [17], the mechanical properties of the high-density collagen sponges can be improved by using EDC which provides cross-linking density higher than that of the DHT treatment. The incorporation of some synthetic biodegradable polymers has been used as a simple and effective method for increasing the mechanical properties of collagen sponges [5,8– 10]. Chen et al. reported that the ultimate tensile strength (10 kPa) and Young's modulus (20 kPa) of the porous collagen sheet under wet conditions were increased to 110 kPa and 1230 kPa, respectively, by incorporating PLGA [5]. Hiraoka et al. reported that the compression modulus of a collagen sponge under wet conditions (below 1 kPa) was increased to approximately 6 kPa by incorporating PGA [10]. However, the use of

synthetic biodegradable polymers has a possible adverse effect on the tissue regeneration because of their low in vivo biodegradability. The significance of our finding is that reinforcement can be achieved without the use of any skeletal materials, i.e., while retaining the bioresorbability of collagen sponges. However, the achieved toughness and stiffness are still far away from being applicable in bone regenerative medicine. The compressive strength and modulus of the collagen sponges reached 8.88 ± 0.93 kPa and 332 ± 39 kPa, respectively, when the concentration of collagen fibrils increased to 10%. On the other hand, the ultimate strength and elastic modulus of human proximal femur were 6.6 and 616 MPa, respectively. It is apparent that the use of bioceramics as inorganic fillers is required to achieve higher mechanical properties. The increase in density of the collagen sponges inevitably decreased the porosity and interconnectivity between the pores. The in vivo bioresorption rate of collagen is high (within several weeks), and consequently the interconnectivity does not seem to be critical for acellular tissue engineering. However, fabrication methods such as porogen leaching methods will be required when the density is further increased. In conclusion, we developed a fabrication technique for collagen sponges with improved mechanical properties involving the use of exhaustive condensation of reconstituted collagen fibrils. This technique does not require the incorporation of any synthetic biodegradable polymers as skeletal materials, and is expected to be utilized for fabrication of collagen-based scaffolds for various tissue engineering applications. Our future research will investigate the potential of the collagen sponges in various orthopedic surgeries in which conventional collagen sponges are not suitable because of their low mechanical properties under wet conditions.

Acknowledgements This research was supported in part by the grant program “Collaborative Development of Innovative Seeds” from the Japan Science and Technology Agency.

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