Biphasic peptide amphiphile nanomatrix embedded with hydroxyapatite nanoparticles for stimulated osteoinductive response

Abstract

Formation of the native bone extracellular matrix (ECM) provides an attractive template for bone tissue engineering. The structural support and biological complexity of bone ECM are provided within a composite microenvironment that consists of an organic fibrous network reinforced by inorganic hydroxyapatite (HA) nanoparticles. Recreating this biphasic assembly, a bone ECM analogous scaffold comprising self-assembling peptide amphiphile (PA) nanofibers and interspersed HA nanoparticles was investigated. PAs were endowed with biomolecular ligand signaling using a synthetically inscribed peptide sequence (i.e., RGDS) and integrated with HA nanoparticles to form a biphasic nanomatrix hydrogel. It was hypothesized the biphasic hydrogel would induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) and improve bone healing as mediated by RGDS ligand signaling within PA nanofibers and embedded HA mineralization source. Viscoelastic stability of the biphasic PA hydrogels was evaluated with different weight concentrations of HA for improved gelation. After demonstrating initial viability, long-term cellularity and osteoinduction of encapsulated hMSCs in different PA hydrogels were studied in vitro. Temporal progression of osteogenic maturation was assessed by gene expression of key markers. A preliminary animal study demonstrated bone healing capacity of the biphasic PA nanomatrix under physiological conditions using a critical size femoral defect rat model. The combination of RGDS ligand signaling and HA nanoparticles within the biphasic PA nanomatrix hydrogel demonstrated the most effective osteoinduction and comparative bone healing response. Therefore, the biphasic PA nanomatrix establishes a well-organized scaffold with increased similarity to natural bone ECM with the prospect for improved bone tissue regeneration.

Figure 1

Macroscopic PA hydrogel images of (a-d) PA-RGDS/PA-S (1:1) and (e-h) PA-S. Biphasic PAs created by self-assembling hydrogels with different HA concentrations: (a,e) 0%, (b,f) 33.3%, (c,g) 50%, and (d,h) 66.7%. HA concentrations (%) calculated as percentage of added HA mass to total hydrogel mass (HA + PA). Scale bar represents 1 mm.

Figure 2

TEM images of biphasic PA hydrogels self-assembled with 50% HA concentration in (a) PA-RGDS/PA-S (1:1) and (b) PA-S. Scale bar represents 100 nm.

Figure 3

Representative Live/Dead fluorescent images of hMSCs encapsulated in (a,b) PA-RGDS/PA-S (1:1) and (c,d) PA-S after 3 days. Both PA hydrogels were imaged with embedded HA concentrations of (a,c) 0% and (b,d) 50%. Viewed under confocal microscopy, viable cells fluoresce green, and dead cells are red. Scale bar represents 10 μm.

Figure 4

Cellularity of hMSCs over 28 days. #PA-RGDS/PA-S (1:1), 0% HA promoted significantly greater cellularity than PA-RGDS, 50% HA and PA-S, 50% HA at Day 0. *PA-RGDS/PA-S (1:1), 0% HA and PA-RGDS/PA-S (1:1), 50% HA exhibited greater cellularity than both PA-S, 0% HA and PA-S, 50% HA per time point. **PA-RGDS/PA-S (1:1), 50% expressed higher cellularity than PA-S, 50% HA at Day 28. ^§Samples significantly increased in cellularity compared to Day 0 (p < 0.05).

Figure 5

Gene expression profile for Runx2 over 28 days. Values expressed as mean ± standard deviation relative to PA-S, 0% HA (dashed line) for each time point. PA-RGDS/PA-S (1:1), 50% HA promoted significantly greater expression than *PA-S, 50% HA on Day 7, **PA-RGDS/PA-S (1:1), 0% HA and PA-S, 50% HA on Day 14, and ^§normalization control of PA-S, 0% HA (p < 0.05).

Figure 6

Gene expression profile for ALP over 28 days. Values expressed as mean ± standard deviation relative to PA-S, 0% HA (dashed line) for each time point. ^§PA-RGDS/PA-S (1:1), 50% HA promoted significantly greater expression than the normalization control of PA-S, 0% HA (p < 0.05).

Figure 7

Gene expression profile for collagen type I over 28 days. Values expressed as mean ± standard deviation relative to PA-S, 0% HA (dashed line) for each time point. PA-RGDS/PA-S (1:1), 50% HA promoted significantly greater expression than *PA-RGDS/PA-S (1:1), 0% HA and PA-S, 50% HA on Day 28 and ^§normalization control of PA-S, 0% HA (p < 0.05).

Figure 8

Gene expression profile for OCN over 28 days. Values expressed as mean ± standard deviation relative to PA-S, 0% HA (dashed line) for each time point. PA-RGDS/PA-S (1:1), 50% HA promoted significantly greater expression than *PA-RGDS/PA-S (1:1), 0% HA and PA-S, 50% HA on Day 14 and **PA-S, 50% HA on Day 28. ^#PA-RGDS/PA-S (1:1), 0% HA expressed more than PA-RGDS/PA-S (1:1), 50% and PA-S, 50% HA on Day 28. ^§Samples greater than the normalization control of PA-S, 0% HA (p < 0.05).

Figure 9

Gene expression profile for MMP-2 over 28 days. Values expressed as mean ± standard deviation relative to PA-S, 0% HA (dashed line) for each time point.

Figure 10

High resolution radiographs taken after (a-c) two and (d-f) four weeks post-operatively. Representative images shown for (a,d) defect only, (b,e) PA-RGDS/PA-S (1:1) hydrogel, and (c,f) biphasic PA-RGDS/PA-S (1:1), 50% HA. All images taken as lateral radiographs of the 6 mm critical size rat femoral defects.

Figure 11

Histological evaluation of 6 mm critical size femoral defects after four weeks using Goldner’s trichrome staining. Representative images shown for (a) defect only, (b) PA-RGDS/PA-S (1:1) hydrogel, 0% HA and (c) biphasic PA-RGDS/PA-S (1:1), 50% HA. Osteoid stains dark pink, and calcified bone tissue appears green.