One-Step Preparation of an Injectable Hydrogel Scaffold System Capable of Sequential Dual-Growth Factor Release to Maximize Bone Regeneration

Abstract

Numerous growth factors are involved in the natural bone healing process, which is precisely controlled in a time- and concentration-dependent manner. Mimicking the secretion pattern of growth factors could be an effective means to maximize the bone regeneration effect. However, achieving the sequential delivery of various growth factors without the use of multiple materials or complex scaffold designs is challenging. Herein, an injectable poly(organophosphazene) hydrogel scaffold (IPS) encapsulating bone morphogenetic protein (BMP)-2 and TGFβ-1 (IPS_BT) is studied to mimic the sequential secretion of growth factors involved in natural bone healing. The IPS_BT system is designed to release TGFβ-1 slowly while retaining BMP-2 for a longer period of time. When IPS_BT is injected in vivo, the hydrogel is replaced by bone tissue. In addition, angiogenic (CD31 and alpha-smooth muscle actin (α-SMA)) and stemness (Nanog and SOX2) markers are highly upregulated in the early stages of bone regeneration. The IPS system developed here has promising applications in tissue engineering because 1) various amounts of the growth factors can be loaded in one step, 2) the release pattern of each growth factor can be controlled via differences in their molecular interactions, and 3) the injected IPS can be degraded and replaced with regenerated bone tissue.

Keywords: angiogenesis; bone healing; growth factors; injectable hydrogels; stem cells.

Figure 1

Schematic of the preparation of injectable poly(organophosphazene) hydrogel scaffolds encapsulating BMP‐2 and TGFβ‐1 and the sequential release of each growth factor.

Figure 2

Temperature‐dependent mechanical properties of IPS encapsulating various BMP‐2 and TGFβ‐1 contents. Storage and loss moduli of a) IPS, b) IPS with BMP2 (20 µg/200 µL), c) IPS with TGFβ‐1 (1 µg/200 µL), and d) IPS with BMP2 (20 µg/200 µL) and TGFβ‐1 (1 µg/200 µL). e) Cryo‐SEM image of IPS before and after thermal crosslinking (Scale bar = 5 µm).

Figure 3

Physical interactions between IPS and the growth factors and the in vitro release of BMP‐2 and TGFβ‐1 from IPS_BT immersed in PBS at 37 °C. a) Particle size of IPS, IPS_B, IPS_T, and IPS_BT with the corresponding PDI values (n = 5). b) Zeta potentials of IPS, IPS_B, IPS_T, and IPS_BT (n = 4). c) Cumulative release profiles of BMP‐2 and TGFβ‐1 over 50 d (n = 3). d) Released, remaining, and undetected amounts of BMP‐2 and TGFβ‐1 determined using ELISA kits (n = 3). Error bars indicate mean ± standard deviation. * p < 0.5, ** p < 0.01, and *** p < 0.001(one‐way analysis of variance with post hoc Tukey's multiple comparison test).

Figure 4

Bone tissue regeneration 4 weeks after the subcutaneous injection of IPS with various BMP‐2 doses (n = 4). a) Micro‐CT images and b) bone volume measurements. Scale bar = 1 mm c) Regenerated tissue in the ectopic region. d) H&E and e) MT staining results of bone tissue regeneration (NB: new bone). Scale bar = 200 µm. Error bars indicate mean ± standard deviation. * p < 0.5, ** p < 0.01, and *** p < 0.001 (one‐way analysis of variance with post hoc Tukey's multiple comparison test).

Figure 5

Bone tissue regeneration 4 weeks after the subcutaneous injection of IPS with various TGFβ‐1 doses (n = 4). a) Micro‐CT images and b) bone volume measurements. Scale bar = 1 mm. c) Regenerated tissue in the ectopic region. d) H&E and e) MT staining results of bone tissue regeneration (NB: new bone). Scale bar = 200 µm. Error bars indicate mean ± standard deviation. * p < 0.5, ** p < 0.01, and *** p < 0.001 (one‐way analysis of variance with post hoc Tukey's multiple comparison test).

Figure 6

Angiogenesis in the IPS system. The angiogenic markers CD31 and αSMA were analyzed after IPS injection. a) Scheme of the in vivo study. Angiogenesis was analyzed 7 d after injection. b) Gene expression levels of CD31 and αSMA at all time points considered (n = 3). c) Western blot results of CD31 and αSMA. d) Quantitative data of the Western blot bands (n = 3). e) Immunofluorescence analysis of the angiogenic markers (green: αSMA, red: CD31, blue: DAPI, yellow arrow: blood vessel). Scale bar = 100 µm. Error bars indicate mean ± standard deviation. * p < 0.5, ** p < 0.01, and *** p < 0.001 (one‐way analysis of variance with post hoc Tukey's multiple comparison test).

Figure 7

Stem cell recruitment in the IPS system. Stemness markers (Nanog, SOX2) were analyzed after IPS injection. a) Scheme of the in vivo study. Stemness was analyzed 14 d after injection. b) Gene expression levels of Nanog and SOX2 at all time points considered (n = 3). c) Western blots of Nanog and SOX2. d) Quantitative data of the Western blot bands (n = 3). e) Immunofluorescence analysis of the stemness markers (green: SOX2, red: Nanog, blue: DAPI). Scale bar = 50 µm. Error bars indicate mean ± standard deviation. * p < 0.5, ** p < 0.01, and *** p < 0.001(one‐way analysis of variance with post hoc Tukey's multiple comparison test).

Figure 8

Orthotopic bone regeneration by IPS encapsulating growth factors. a) Micro‐CT images of calcified bone. Scale bar = 5 mm. b) Regenerated bone volume quantitation (n = 3). c) Photographs of extracted calvarial tissue collected 4 weeks after IPS injection. d) H&E and e) MT staining results indicating new bone regeneration. Scale bar = 200 µm. Error bars indicate mean ± standard deviation. * p < 0.5, ** p < 0.01, and *** p < 0.001(one‐way analysis of variance with post hoc Tukey's multiple comparison test).