Addressing the Osteoporosis Problem-Multifunctional Injectable Hybrid Materials for Controlling Local Bone Tissue Remodeling

Abstract

Novel multifunctional biomimetic injectable hybrid systems were synthesized. The physicochemical as well as biological in vitro and in vivo tests demonstrated that they are promising candidates for bone tissue regeneration. The hybrids are composed of a biopolymeric collagen/chitosan/hyaluronic acid matrix and amine group-functionalized silica particles decorated with apatite to which the alendronate molecules were coordinated. The components of these systems were integrated and stabilized by cross-linking with genipin, a compound of natural origin. They can be precisely injected into the diseased tissue in the form of a viscous sol or a partially cross-linked hydrogel, where they can serve as scaffolds for locally controlled bone tissue regeneration/remodeling by supporting the osteoblast formation/proliferation and maintaining the optimal osteoclast level. These materials lack systemic toxicity. They can be particularly useful for the repair of small osteoporotic bone defects.

Keywords: biocompatibility in vivo; biopolymers; bone tissue engineering; injectable hydrogels; osteoporosis; silica−apatite-based alendronate carriers.

Figure 1

Scheme of SiO₂–Ap–ALN particle synthesis (SiO₂–NH₂—silica particles functionalized with amine groups; Ap—apatite formed on the surface of silica particles; and ALN—sodium alendronate).

Figure 2

(A) SEM micrographs, (B) XRD pattern, and (C) TG profiles obtained in an inert atmosphere (argon) for silica particles covered with Ap (SiO₂–Ap) and ALN-attached (SiO₂–Ap–ALN).

Figure 3

(A) SR for the pristine hydrogel and hybrid materials with the sodium ALN carrier incubated in PBS for 24 h. * indicates statistical significance when compared with ColChHA_mod C1. (B) Contact angle values (deg) measured on surfaces of tested materials. (C) Changes in the pristine hydrogel and hybrid material weight during 144 h of enzymatic degradation. Statistical analysis was carried out by comparing all types of materials at the same time of degradation. * indicates statistical significance when compared with ColChHA_mod after 24 h of experiment, # indicates statistical significance when compared with ColChHA_mod after 72 h of experiment, and ^ indicates statistical significance when compared with ColChHA_mod after 144 h of experiment. (D) Values of the storage modulus (G′) measured in 10, 35, and 70 min after starting the experiment are presented on a logarithmic scale. Statistical significance was calculated using Student’s t-test. A comparison between two means was analyzed with a statistical significance level set at p < 0.05; * indicates statistical significance when compared with ColChHA_mod after 70 min and # indicates statistical significance when compared with ColChHA_mod after 70 min.

Figure 4

Cumulative release of ALN from hybrid material ColChHA_mod C1.

Figure 5

SEM images of the surface of pristine hydrogel and hybrid materials with the sodium ALN carrier after 3 and 5 days of incubation in SBF. The Ca/P ratios determined by EDS analyses.

Figure 6

(A) SEM images of the MG-63 cell morphology on the surface of the pristine hydrogel and hybrid materials with the sodium ALN carrier. (B) Distributions of the spread area for MG-63 cells cultured on the pristine hydrogel and hybrid materials with the sodium ALN carrier. The area of cells was calculated employing ImageJ software. Boxes represent interquartile range and median. (C) Cell number and (D) ALP activity of MG-63 cells grown on the surface of the materials studied on days 1, 3, and 7 of the culturing. Statistical significance was calculated using Student’s t-test. In (C), % indicates statistical significance when compared with ColChHA_mod on day 3 and $ indicates statistical significance when compared with ColChHA_mod on day 7. In (D), * indicates statistical significance when compared with control on day 3, ** indicates statistical significance when compared with control on day 7, and # indicates statistical significance when compared with ColChHA_mod C1 on day 3. Cells cultured on the tissue culture plate were considered as control. (E) Cell number of J774A.1 cells grown on the surface of the materials studied on days 1, 3, and 7 of culturing. Statistical significance was calculated using Student’s t-test. % indicates statistical significance when compared with ColChHA_mod on day 1.

Figure 7

(A) Fragments of isolated skin with hydrogel-based materials. Animals were subcutaneously injected with PBS or with hydrogels and euthanized after various periods. Skin fragments containing hydrogels were isolated, photographed, and then processed for further analyses. Representative pictures taken after various periods show changes in the hydrogels’ size, structure, and color, and indicate hydrogel degradation (n = 10). (B) Graph shows a decrease in the hydrogel volume observed after various periods followed by material injection. Each point on the graph represents an individual animal; each group’s line represents mean ± SD (n = 5). (C) Representative picture of ColChHAmodC1 isolated from mice skin after 60 days postinjection taken using a scanning electron microscope. The white circle indicates HAp aggregates as determined by EDS analysis. The white arrow points out the blood vessel in the hydrogel.

Figure 8

Blood hematology analyses performed for mice exposed to hydrogels. Animals injected with PBS or with materials studied were euthanized after various periods. Blood was taken before euthanasia from the facial vein. Bars for each group represent the mean ± SD (n = 7).

Figure 9

Biochemical serum analyses performed for mice exposed to hydrogels. Animals were subcutaneously injected with PBS or with materials studied and euthanized after various periods. Blood was taken after euthanasia by cardiac puncture, and sera were isolated. Each point on the graph represents an individual animal; each group’s line represents mean ± SD (n = 7).

Figure 10

Cytokine detection in sera after materials administration. Animals were subcutaneously injected with PBS or with hydrogels and euthanized after various periods. Blood was taken after euthanasia by cardiac puncture, and sera were isolated. Each point on the graph represents an individual animal; each group’s line represents mean ± SD. Only values that were within the detection range were plot in the graph. For some cytokines, the indicated concentration was below the detection level (but all were analyzed according to the LEGENDplex 13-plex Mouse inflammatory panel). Analyzed n = 10, but the graph has a notable different value of n = 2–10.

Figure 11

Hydrogel ColChHAmod stained with Masson’s trichrome. Animals were subcutaneously injected with PBS or with materials and euthanized after various periods. Skin fragments containing hydrogels were isolated, processed, and stained with Masson’s trichrome. The white arrow indicates neutrophils and dark blue arrow indicates macrophages.

Figure 12

Hydrogel ColChHAmod C1 stained with Masson’s trichrome. Animals were subcutaneously injected with PBS or with materials and euthanized after various periods. Skin fragments containing hydrogels were isolated, processed, and stained with Masson’s trichrome to visualize hydrogel infiltration by host cells and detect Col. The square represents an area that is shown at a larger magnification. The white arrow indicates neutrophils and the dark blue arrow indicates macrophages.