Injectable nanohydroxyapatite-chitosan-gelatin micro-scaffolds induce regeneration of knee subchondral bone lesions

B Wang, W Liu, D Xing, R Li, C Lv, Y Li, X Yan, Y Ke, Y Xu, Y Du, J Lin, B Wang, W Liu, D Xing, R Li, C Lv, Y Li, X Yan, Y Ke, Y Xu, Y Du, J Lin

Abstract

Subchondral bone has been identified as an attractive target for KOA. To determine whether a minimally invasive micro-scaffolds could be used to induce regeneration of knee subchondral bone lesions, and to examine the protective effect of subchondral bone regeneration on upper cartilage, a ready-to-use injectable treatment with nanohydroxyapatite-chitosan-gelatin micro-scaffolds (HaCGMs) is proposed. Human-infrapatellar-fat-pad-derived adipose stem cells (IPFP-ASCs) were used as a cellular model to examine the osteo-inductivity and biocompatibility of HaCGMs, which were feasibly obtained with potency for multi-potential differentiations. Furthermore, a subchondral bone lesion model was developed to mimic the necrotic region removing performed by surgeons before sequestrectomy. HaCGMs were injected into the model to induce regeneration of subchondral bone. HaCGMs exhibited desirable swelling ratios, porosity, stiffness, and bioactivity and allowed cellular infiltration. Eight weeks after treatment, assessment via X-ray imaging, micro-CT imaging, and histological analysis revealed that rabbits treated with HaCGMs had better subchondral bone regeneration than those not treated. Interestingly, rabbits in the HaCGM treatment group also exhibited improved reservation of upper cartilage compared to those in other groups, as shown by safranin O-fast green staining. Present study provides an in-depth demonstration of injectable HaCGM-based regenerative therapy, which may provide an attractive alternative strategy for treating KOA.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Schematic illustration of overall research design. (A) Articular structures that are affected between normal and osteoarthritis tissues, including damaged cartilage, weakened muscles, inflamed synovium, and subchondroal bone cysts. (B) Schematic showing the subchondral bone lesion model that was established to mimic the reparative progress using injectable HaCGMs to fill a cavity. HaCGMs stained red solely for visualization purpose were homogeneously suspended in 20% gelatin solution. (C) Regeneration of subchondral bone lays the foundation for the upper cartilage. We got permission from Xiuxiu Wang for publication of the image.
Figure 2
Figure 2
Scanning electron microscopy (SEM) images, pore size statistics and characteristics of HaCGMs: (A) SEM images of HaCGMs with HAp (0%, 1%, 3% and 5%) showing interconnected macroporous structures. (B) The pore size distribution of HaCGMs with HAp (0%, 1%, 3% and 5%). (C) Live/dead staining with Calcein AM/PI showed that IPFP-ASCs seeded in HaCGMs with 3% HAp have a higher proliferative capacity. (D) Strain-stress curves for HaCGMs with HAp (0%, 1%, 3% and 5%) subjected to compression tests. (E–F) The porosity and swelling ratio of HaCGMs with HAp (0%, 3%). The data are the means ± SEM; n = 4.
Figure 3
Figure 3
Degradation of HaCGMs. (A)Images showing the in vitro degradation rate of HaCGMs with 3% HAp and gelatin micro-scaffolds. (B) Schematic showing the in vivo degradation rate of HaCGMs with 3% HAp after subcutaneous injection. (C–D) Macroscopic view of HaCGMs after 2 weeks and 8 weeks. Note that dense and organized blood vessels were found to form around reddish fibrous capsules at the injection sites. H&E staining showing endogenous cell loading around or inside HaCGMs. M = Microscaffold. We got permission from Xiuxiu Wang for publication of the image.
Figure 4
Figure 4
Characterization of infrapatellar-fat-pad-derived stem cells (IPFP-ASCs). (A) The location of the infrapatellar fat pad. IPFP-ASCs and bone marrow stem cells have flat polygonal morphology at the 3rd passages under light microscope. (B) The multi-lineage differentiation ability of IPFP-ASCs was assessed by staining with appropriate specific reagents. Positive staining was observed following osteogenic, chondrogenic, or adipogenic differentiation using specific induction media. (C) Flow cytometry analysis of IPFP-ASCs revealed positive markers (CD 73, CD 90, CD 105). (D) IPFP-ASCs were encapsulated in HaCGMs and cultured in osteogenic induction medium, with gelatin microcryogels and 2-dimension (2D) culture as controls. ELISA analysis revealed higher ALP activity produced by cells cultured in HaCGMs on day 14 compared to day 7. Quantitative RT-PCR showed that the gene expression of osteoinductive markers, such as alkaline phosphatase (ALP), type I collagen (COL1), and RUNX2, were up-regulated. The data are expressed as the relative gene expression normalized to that of the housekeeping gene. The data are the means ± SEM; n = 4.
Figure 5
Figure 5
In vivo therapeutic effect of injectable HaCGMs for subchondral bone lesion. (A) HaCGMs were injected in a rabbit model of subchondral bone lesion, which imitated the actual necrosis removing of subchondral bone cysts. (B) Photographic images showing the details of subchondral bone lesions. (C) Full-body roentgenogram showing no fibrosis agent in the proximal metaphysis of the tibia or in the lungs at the same time. Lateral roentgenogram of the proximal tibia showed that rabbits treated with the HaCGMs had better regeneration. (D) Evaluation of the Lane-Sandhu radiological score. The data are shown as the means ± SEM; n = 6.
Figure 6
Figure 6
Assessment of subchondral bone lesion regeneration by micro-CT analysis. (A) The tibias surface of rabbits was evaluated by 3D reconstruction based on micro-CT images. (B–C) micro-CT was used to generated 3D reconstruction of subchondral lesion that showed regeneration differences among the different groups. (D) The parameters of microarchitecture (BV/TV, Tb. N, Tb. T, and Tb. S) were measured in the trabecular bone of the proximal tibia (1–2 mm distal to the proximal physis). HaCGM treatment induced a significant increase in BV/TV and Tb. N compared with other treatments (p < 0.05). No difference was observed in Tb. T and Tb. S. BV/TV = bone volume/total volume; Tb. N = trabecular number; Tb. T = trabecular thickness; Tb. S = trabecular spacing. The data are shown as the means ± SEM; n = 6.
Figure 7
Figure 7
Histopathological analysis of subchondral bone regeneration. (A–B) Representative histological H&E and Masson trichrome staining of longitudinal sections of tibia at 8 weeks post-surgery. Diagram showing the relative trabecular area. Data are shown as the means ± SEM; n = 9.
Figure 8
Figure 8
Histological analysis of the protective role of subchondral bone regeneration with regard to the upper cartilage. (A) Representative histological safranin-O & fast green staining of longitudinal sections of tibia at different magnifications. (B) Diagram showing the results of histological grading evaluated at 8 weeks post-surgery according to OARSI scores and HSS scores. The data are shown as the means ± SEM; n = 9.

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