Large animal in vivo evaluation of a binary blend polymer scaffold for skeletal tissue-engineering strategies; translational issues

Abstract

Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l-lactic acid)-poly(ε-caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow-derived skeletal stem cells (SSCs) were implanted into mid-shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro-CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical-sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale-up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale-up this stem cell/binary blend polymer scaffold. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

Keywords: binary blend; bone regeneration; polymer; scaffold; skeletal stem cell.

Figure 1

Binary polymer scaffold following processing: note the longitudinal intramedullary canal; scale bar = 10 mm. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Tibial segmental defect operative procedure. Following skin preparation and draping: (a) an anteromedial approach was used to access the diaphyseal portion of the right tibia; (b) the periosteum was carefully and entirely removed around the site of the proposed ostectomy; (c) the proprietary jig was secured against the bone and the 35 mm ostectomy was marked on the tibia, using diathermy; (d) six 4 mm diameter holes were drilled through the jig guides; and (e) Schanz screws were inserted in a standardized order; (f) the jig was removed and the external fixator was secured in place prior to forming the ostectomy at the pre‐marked site, using an electric reciprocating sagittal saw; (g) the segment of bone was removed, along with any remaining periosteum; (h) polymer scaffold inserted, with or without SSCs; (i) appearance following closure. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Cubes of tissue/polymer were taken from the indicated regions for histological analysis: (a) the interface between the proximal cut end of tibia and the polymer scaffold; (b) an area on the surface of the mid‐section of the scaffold; (c) an area on the inner face of the scaffold mid‐section

Figure 4

SEM images of polymer scaffold, demonstrating multiple pores of varying dimensions necessary for rapid cellular infiltration

Figure 5

Alcian blue penetration test: (a) the polymer was suspended in a perforated specimen container and the proximal end ‘sealed’ with histology wax before addition of Alcian blue; (b) polymer scaffold following removal of wax – the blue dye is visible throughout the sample; (c) the scaffold was dissected; (d) cross‐sections of the scaffold from proximal (left) to distal (right), demonstrating even penetration of dye throughout the specimen. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

The typical pattern of new bone formation in each group. In the empty defects (3), minimal osteogenesis formed into a conical pattern, mainly from the proximal cortices; in the scaffold‐alone group (5), increased bone formation was evident from both the proximal and distal bone ends, that appeared more evenly throughout the structure of the scaffold polymer; in the scaffold and cells group (11), most bone formation occurred within the central cannulation of the scaffold

Figure 7

Quantitative μCT analysis at 12 weeks post‐operation: there is minimal regenerative activity in the empty defect specimens; some bone formation is seen in both scaffold groups, although there is no certain difference in the group with added SSCs

Figure 8

Quantitative μCT analysis of new bone formation after 12 weeks of incubation; error bars, SD; ns, not significant

Figure 9

Results of mechanical testing for the sheep tibiae under torsional compression, demonstrating: (a) maximum torque; and (b) maximum shear stress before failure; control refers to the contralateral intact tibia; error bars, SD

Figure 10

Macroscopic specimen analysis following mechanical testing. Regions of interest in each image show the proximal diaphysis (left), the distal diaphysis (right) and defect containing no scaffold (1–4), scaffold alone (5–8) or scaffold with cells (9–12). Failure occurred through the scaffold itself in three of the four specimens in each scaffold group; however, failure occurred at the distal scaffold–diaphysis interface in one specimen of each scaffold group (arrows in 8 and 11). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 11

Macroscopic image of a tibia treated in the scaffold + SSCs group (specimen 9 in Figure 10). The polymer scaffold in this case was largely fragmented following mechanical testing and has been carefully removed to reveal a central bridge of new tissue formation within the medullary cavity of the scaffold; note full continuity between the proximal and distal diaphyseal segments. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 12

A/S histological analysis of the ovine segmental tibial defect model after 12 weeks in vivo incubation. Only the scaffold groups are displayed, as insufficient tissue was formed within the empty defects. In region A (bone–scaffold interface), there was significant infiltration of new tissue into the scaffold, as demonstrated by red staining of collagen type I. In region B (near the surface of the mid‐section of the scaffold), new osseous tissue was seen in both scaffold groups, with deep penetration of tissue into the porous network. Region C (within the inner face of the scaffold mid‐section) showed no new osseous tissue formation in either scaffold group; the polymer in this region remained intact, with some surrounding cells but no new bone formation; *polymer; scale bars = 100 µm. [Colour figure can be viewed at wileyonlinelibrary.com]