The effect of mesenchymal stem cells delivered via hydrogel-based tissue engineered periosteum on bone allograft healing

Abstract

Allografts remain the clinical "gold standard" for treatment of critical sized bone defects despite minimal engraftment and ∼60% long-term failure rates. Therefore, the development of strategies to improve allograft healing and integration are necessary. The periosteum and its associated stem cell population, which are lacking in allografts, coordinate autograft healing. Herein we utilized hydrolytically degradable hydrogels to transplant and localize mesenchymal stem cells (MSCs) to allograft surfaces, creating a periosteum mimetic, termed a 'tissue engineered periosteum'. Our results demonstrated that this tissue engineering approach resulted in increased graft vascularization (∼2.4-fold), endochondral bone formation (∼2.8-fold), and biomechanical strength (1.8-fold), as compared to untreated allografts, over 16 weeks of healing. Despite this enhancement in healing, the process of endochondral ossification was delayed compared to autografts, requiring further modifications for this approach to be clinically acceptable. However, this bottom-up biomaterials approach, the engineered periosteum, can be augmented with alternative cell types, matrix cues, growth factors, and/or other small molecule drugs to expedite the process of ossification.

Keywords: Bone allografts; Hydrogels; Mesenchymal stem cells; Periosteum; Regenerative medicine; Tissue engineering.

Figure 1

Scheme representing the tissue engineered approach to enhance allograft healing. mMSCs were added to poly(ethylene glycol) macromer solutions (A) and custom molds were used to form hydrogel-cell constructs around decellularized allografts (e.g., tissue engineered periosteum) (B). Encapsulated cells remained >95% viable as illustrated by the live/dead image (of GFP⁻ mMSCs; calcein AM (green = live cells), ethidium homodimer (red – dead cells)) 24 hr after encapsulation (C).

Figure 2

Allografts were modified with PEG hydrogels encapsulating GFP⁺ mMSCs and implanted into mouse femurs with 5 mm segmental defects (A). In vivo MSC persistence at the allograft surface was followed using live animal fluorescent imaging (B). Compared to control allografts directly seeded with GFP⁺ mMSCs, T.E. periosteum modified allografts exhibited increased normalized MSC localization through ~12 days as in agreement with hydrogel degradation kinetics (C; Fig. S2) (n=5; error bars represent standard error of the mean; p-value of <0.05 indicates significance compared to directly seeded (*)).

Figure 3

Micro-computed tomography scans were used to assess in vivo graft vascularization (A). Quantification revealed tissue engineered periosteum modified allografts exhibited enhanced vascular volume as compared to allograft controls (B) (n=5; error bars represent standard error of the mean; p-value of <0.05 indicates significance compared to allograft (*) or autograft (#)).

Figure 4

Micro-computed tomography scans were used to assess in vivo bone callus formation. Reconstructed graft calluses show both full intact scans and sagital cut views (A). Subsequent quantification revealed increased bone callus volume in tissue engineered periosteum modified allografts as compared to allograft only controls (B) (n=5; error bars represent standard error of the mean; p-value of <0.05 indicates significance compared to allograft (*) or autograft (#)).

Figure 5

Histological analysis of graft sections revealed extensive cortical bone resorption (black arrows) and endochondral bone formation (black triangles) bridging the defect in tissue engineered periosteum modified allografts as compared to untreated allograft controls (A; i–ix). The presence of glycosaminoglycans and proteoglycans was detected via alcian blue staining (blue), and bone and surrounding soft tissue was stained with orange G (pink). Closer examination of the bridging callus formed across tissue engineered periosteum modified allografts (B (10× magnification of black triangles; i–iii)) revealed a hypertrophic condensation phenotype (B (40× magnification of black dashed squares; iv–vi)) consistent with chondrogenic differentiation of the transplanted GFP⁺ mMSC population (B (40× magnification of black dashed squares; vii–ix)). Furthermore, GFP⁺ mMSC colocalized to regions of cartilaginous matrix (alcian blue staining; iv–vi) suggesting that transplanted MSCs directly contributed to endochondral mediated bone formation.

Figure 6

Histomorphometric analysis of alcian blue (blue, glycosaminoglycans/proteoglycans) orange G (pink, bone/soft tissue) stained graft sections (Fig. 5A) revealed a significant increase in total callus (A), total mesenchyme callus (B), total cartilage callus (C), and total woven bone callus (D) area in tissue engineered periosteum modified allografts as compared to allograft only controls. (n=8–10; error bars represent standard error of the mean; p-value of <0.05 indicates significance compared to allograft (*) or autograft (#)).

Figure 7

Maximal torsion strength of tissue engineered periosteum modified allograft-host union was significantly increased over untreated allograft controls 16 weeks post-implantation (A) (n=10). Union ratios calculated for graft-host connectivity followed the same trend as those found via histomorphometric analysis of total woven bone callus area (Fig. 6D) and torsional biomechanics (A). While statistical significance was not achieved, qualitative and quantitative analysis revealed increased graft-host union (regions of white) over the healing time course for tissue engineered periosteum modified allografts as compared to untreated allograft controls (B) (n=6; error bars represent standard error of the mean; p-value of <0.05 indicates significance compared to intact femur (*), autograft (#), or allograft ($)).