Effects of bio-inspired bioglass/collagen/magnesium composites on bone repair

Abstract

Bone disorders and fractures affect millions of people worldwide. In critical situations, proper healing may not happen which may lead to non-union fractures. Consequently, the development of new clinical approaches is necessary. In this context, the present work developed bioglass/collagen/magnesium composites in order to mimic natural bone, toward the enhancement of material mechanical properties, but keeping bioactivity and performance of bioglass and collagen. So, the aim of this investigation was to explore the biological outcomes of bioglass/collagen/magnesium composites over bone repair, using tibial defect in Wistar rats. For this purpose, histopathological, morphometrical and immunohistochemical analyses, as well as mechanical test, were performed after 15 and 45 days of the composites implantation in rats. Histopathological assessment indicated that the biocompatible composites degraded over time, leading to a progressive new bone formation from the border to the core of the defect. Immunohistochemistry showed that all groups presented runx-2 and Rank-L immunostaining, with improved runx-2 labeling for bioglass/magnesium compared to plain bioglass 45 days post-implantation. Mechanical tests of the tibiae indicated good mechanical properties of these Mg-based materials, but no statistical differences among groups were found. Additional studies should be done to confirm the outcomes of bioglass/collagen/magnesium on bone restoration, using different animal models and new balanced amounts of Mg (3 wt%< Mg < 5 wt%), as well as compromised, long-term and critical bone defect conditions.

Keywords

Bioglass collagen magnesium composites bone repair

Introduction

Millions of people are affected worldwide by bone disorders and clinical event of bone fractures annually. These events are generally related do aging, traumas, or disease.^1,2 The bone tissue is able to recover by itself in terms of mechanics and structure, even though this restoration being very complex.^3–5 Nevertheless, in critical contexts, like bulky bone defects, lack of nutrients or mechanical impairment, proper healing may not happen, leading to atypical consolidation or non-union fractures.^5,6

Considering this matter, the development of clinical approaches for inducing bone repair toward proper consolidation is needed. Following this line, biomaterials for bone grafting are very promising and appealing, since they are capable of stimulating cell differentiation and neoformed bone deposition, enhancing tissue restoration.^7–10 The bioactive glasses (BGs) are one of the main class of biomaterials, presenting high bioactivity and osteogenic properties.^11–13 BGs, in close contact with tissues and fluids, develop a hydroxycarbonate apatite film, forming a strong tissue/implant binding, which mimics the natural interface in the restoration of bone.^11,13 Previous work confirmed that BG stimulates the proliferation and differentiation of osteoprogenitor cells into osteoblasts, accelerating the neoformed bone deposition and ingrowth.^11,14,15

Despite the known osteogenic properties of BG, these may not be adequate to induce consolidation in complex and critical situation.^13,14 Therefore, the combination of materials from different sources and classes, known as composites, has been shown a viable and promising approach.^16–19 In this context, the combination of an inorganic material, like BG, to an organic one, such as collagen (Col), may be a better alternative to mimic the structure and composition of the bone tissue,^18,20,21 constituting a biomaterial with enhanced biological performance.

It is already verified and known that Col scaffolds are capable of upregulating bone osteopontin, bone morphogenetic proteins (BMPs), and bone sialoprotein, inducing bone cell differentiation and proliferation in models of bone defects.^20,22 Previous in vitro studies demonstrated that Col-containing composites upregulated alkaline phosphatase of osteoblastic cell.²³ Recent researches showed that Col scaffolds induced increased expression of runx-2 and osteocalcin in osteoblastic cell lineages, enhanced endochondral ossification, and improved bone quality in an in vivo investigation.^22,24

In spite of the promising activity of BG and Col composites over bone tissue stimulation, they still present poor mechanical properties, mainly for utilization in load-bearing applications.²⁵ In order to overcome this restriction, the improvement of the material mechanics by using a metallic constituent, as magnesium (Mg), seems to be a feasible approach. Mg²⁺ is the fourth most abundant cation in the human organism and has been a promising element for manufacturing biomaterials composites, with better mechanical properties.²⁶ In this context, studies that explored Mg/Ca alloys²⁶ and BG/Mg-based materials²⁷ demonstrated improved mechanical properties, degradation, and low corrosion; additionally, these biomaterials stimulated the proliferation and differentiation of osteoblastic cells,^26,27 being suitable for bone tissue healing. In a more recent investigation of our research group, Gabbai-Armelin et al. successfully incorporated Mg into BG/Col and showed that the BG/Col/Mg composites presented improved mechanical properties and were biocompatible, being a feasible alternative to be utilized for bone tissue engineering applications.²⁸

Due to the rising necessities in developing suitable biomaterials for bone grafts, we hypothesized that the combination of Mg with BG/Col composites would enhance material mechanical properties, keeping the bioactivity and performance of BG and Col. So, the objective of this research was to investigate the biological outcomes of BG/Col/Mg composites over bone repair, utilizing tibial defect in rats.

Materials and methods

Materials

BG (SiO₂-CaO-Na₂O-P₂O₅ system)^11,29 was provided by Nuclear and Energy Research Institute (125–250 µm; IPEN, São Paulo, Brazil). Tendon bovine Col type I was provided by United States Biological (<500 µm; US Biological Life Sciences, MA, USA) and magnesium powder (74–105 µm), by Alfa Aesar (MA, USA; purity: 99.6%).

Poly (methyl methacrylate) (PMMA, particle size: 15 µm) and methyl methacrylate (MMA, purity: 99.09%) were provided by VIPI Dental Products (Pirassununga, São Paulo, Brazil). Carboxymethyl cellulose (CMC), density 1.59 g/cm³, was provided by Sigma Aldrich (St. Louis, MO). The polymer and monomer were used solely to combine all the tested materials, i.e., BG, Col, and Mg. It is well known that PMMA is biocompatible and inert.^30,31

Preparation of BG/Col/Mg composites

For preparing BG/Col/Mg composites, the materials (i.e., PMMA, MMA, BG, Col, Mg, CMC and distilled H₂O) were added at different ratios according to each group (Table 1). Furthermore, the amounts of BG, Col, and Mg in the BG/Col/Mg composites, taking into account only the weight (wt%) of these materials, are shown in Table 2.

Table 1.

Experimental formulations of BG/Col/Mg composites.

Groups	PMMA (wt%)	MMA (wt%)	Bioglass (wt%)	Collagen (wt%)	Magnesium (wt%)	CMC (wt%)	Water (wt%)
BG	13.14	26.28	26.72	0.00	0.00	2.39	31.47
BG/Mg	13.28	26.57	22.97	0.00	2.94	2.42	31.83
BG/Col	13.64	27.28	19.42	4.51	0.00	2.48	32.68
BG/Col/Mg	13.80	27.59	15.43	4.56	3.05	2.51	33.05

BG: bioglass; CMC: carboxymethyl cellulose; Col: collagen; MMA: methyl methacrylate; PMMA: poly (methyl methacrylate).

Table 2.

Amounts of BG, Col, and Mg in the BG/Col/Mg composites, considering only the wt% of these components.

Groups	Bioglass (wt%)	Collagen (wt%)	Magnesium (wt%)
BG	100	0	0
BG/Mg	89	0	11
BG/Col	81	19	0
BG/Col/Mg	67	20	13

BG: bioglass; Col: collagen.

CMC was used as the porogenic agent^32,33 and the amount utilized for this constituent in each composite is toward inducing porosity of 60%.^34,35 The quantity of PMMA used was the lowest one (reached by tests) practicable to combine the materials. Briefly, all the powder materials were weighed and mixed in a silicone container using spatula. Afterwards, water was added, and the blend was mixed again. Lastly, the MMA monomer was added and mixed in order to start the crosslink. Then, the mixture was quickly transferred to a silicon mold of 3 mm diameter × 1 mm height. Later, the molds were closed and submitted to a pressure air chamber at 0.6 MPa for 30 min. Subsequently, the unsealed molds were vacuum dried (10⁻³ Torr) for 15 min and the composites were set to dry at room temperature.

Experimental design

Eighty male Wistar rats (12 weeks, weighing 300–350 g) were used in this study. They were kept under controlled temperature (24 ± 2°C), dark/light periods of 12 h, with commercial diet, and water ad libitum. Handling and surgical procedures of the animals were directed according to the protocols approved by Animal Care Committee guidelines of the UNIFESP (CEUA n. 2543180915). Animals were divided into four groups: (i) bioglass (BG); (ii) bioglass/collagen (BG/Col); (iii) bioglass/magnesium (BG/Mg), and (iv) bioglass/collagen/magnesium (BG/Col/Mg). These experimental groups were separated into two different sub-groups (n = 10) and euthanized 15 and 45 days after implantation. As described in the following sections, a non-critical size bone defect was done on both tibiae.

Surgical procedures

Surgery was performed under sterile conditions and general anesthesia was induced by intraperitoneal injection of 8 mg∕kg xylazine (Anasedan; Sespo Industry and Trade Ltda, Jacareí, Sao Paulo, Brazil) and 80 mg∕kg ketamine (Dopalen; Sespo Industry and Trade Ltda), associated with 1 mg/kg acepromazine (Dopalen), and 0.05 mg/kg fentanyl (Dopalen). After anesthesia, bilateral non-critical size bone defects (3 mm Ø) were created, utilizing a motorized drill (Beltec®, Araraquara, Sao Paulo, Brazil) and under abundant saline solution irrigation, at the superior third of the tibia (10 mm distal of the knee joint). The composites were positioned in the defect randomly. Afterwards, the wound and skin were closed with resorbable Vicryl® 5-0 (Johnson&Johnson, St.Stevens-Woluwe, Belgium). Post-surgery, all animals received 2 mg/kg meloxicam (Maxicam®, Ourofino, Osasco, Sao Paulo, Brazil) and 60 mg/kg cephalothin (Keflin, Neutro®, Ely Lilly, Sao Paulo, Brazil), for five successive days. Animals were observed daily. Fifteen and 45 days post-implantation, the rats were euthanized by anesthesia overdose and the specimens were collected.

Histopathological analysis

In the histopathological and immunohistochemical analyses, the right tibiae were detached, fixed in 10% buffer formalin (Merck, Darmstadt, Germany) for 24 h, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) (Merck) and embedded in paraffin blocks. Thin sections (5 µm) were prepared using a microtome (Leica Microsystems SP 1600, Nussloch, Germany). Two sections of each specimen were stained with hematoxylin and eosin (Merck) and examined by light microscopy (Leica Microsystems AG, Wetzlar, Darmstadt, Germany).^36–38 The bone defect area was qualitatively evaluated considering inflammatory process, granulation tissue, neoformed bone, and biomaterial degradation. The analysis was performed in a blinded way (PRGA and HWK).

The histomorphometric analysis was done by using a Zeiss microscope (Carl Zeiss Vision GmbH, Germany) and the semiautomatic image-analyzing computer program Osteomeasure (Osteometrics, Inc., Atlanta, GA). Measurements were performed at three standardized fields inside the defect site (region of interest) at magnification of 100×. The indices obtained were: bone volume as a percentage of tissue volume, i.e. bone formation (BV/TV, %), and osteoblastic surface as a percentage of bone surface (ObS/BS, %), which were reported according to the standard nomenclature recommended by the American Society of Bone and Mineral Research.³⁹ The measurements were performed by a single observer (PRGA).

Immunohistochemistry

The immunostaining of runt-related transcription factor-2 (runx-2) and activator of nuclear factor kappa-B ligand (Rank-L) was assessed as defined earlier,^36,37 applying the streptavidin–biotin-peroxidase technique. The immunostainings of runx-2 and Rank-L were assessed qualitatively (immunomarkers presence and location) and quantitatively utilizing a light microscopy (Leica Microsystems AG, Wetzlar), following a previously described scoring scale from 1 to 4 (1 = absent, 2 = weak, 3 = moderate, and 4 = intense).³⁶ The analysis was done in a blinded way (PRGA and KRF).

Mechanical test

Biomechanical properties of the left tibia were determined by a three-point bending test with a 1 kN load (3340 Series Single Column Systems, Instron, Norwood, MA). Tibiae were positioned on a 3.8-cm metal device, which provided a 1.8-cm distance between the two supports. The load cell was perpendicularly placed in the posterior-anterior direction at the exact location of the bone defect. A 5-N pre-load was applied to avoid sample sliding. The bending force was applied at a constant deformation rate of 0.5 cm/min till fracture occurrence. From the load–deformation curve, the maximum load at failure (N) was acquired.

Statistical analysis

Data were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA). Shapiro–Wilk normality test was used to check distribution. Kruskal–Wallis test and Dunn post hoc were used for nonparametric data. One-way analysis of variance and Tukey multiple comparisons post-tests were used for parametric data. Differences were considered significant at p ≤ 0.05.

Results

Histopathological analysis

Fifteen days

After 15 days of implantation, for BG and BG/Col the defect line still could be seen. Moreover, the defect area was filled mostly with granulation tissue and with some biomaterial particles. Newly formed bone was observed mostly in the periphery of the defect for both groups (Figure 1(a) and (b)).

For BG/Mg and BG/Mg/Col samples, material was observed filling the defect region, with granulation tissue (more evident in BG/Col/Mg). Both groups showed new formed bone mainly in the border of the defect (Figure 1(c) and (d)). The defect line was present and medullary tissue was noticed in some Mg-based samples.

The histological findings at day 15 were very similar when comparing groups without and with Mg. It is worth mentioning that BG/Col/Mg presented the most evident granulation tissue among groups (Figure 1(a)–(d)).

Forty-five days

Forty-five days post-implantation, BG and BG/Col showed progressive bone formation in regions which were previously filled with the material (Figure 2(a) and (b)). Medullary tissue could be observed in a few samples and the defect line was less evident compared to the earlier period. Some granulation tissue was noted, mainly in the core of the defect among the remaining particles of the biomaterial. The amount of granulation tissue was more evident for BG/Col compared to BG.

Regarding BG/Mg, a more evident bone formation was noted compared to the previous period. Biomaterial particles could still be noticed, mainly in the central region of the defect for both BG/Mg and BG/Col/Mg (Figure 2(c) and (d)). In some samples, remaining granulation tissue was found among the biomaterial, as well as medullary tissue. Interestingly, BG/Col/Mg showed a more evident granulation tissue all over the defect, and less material degradation and bone formation compared to BG/Mg.

Histomorphometric analysis

The histomorphometry indicated that %BV/TV for BG, BG/Col, BG/Mg, and BG/Col/Mg as ∼27%, 22%, 38%, and 15%, respectively, at day 15. Similar statistical findings were observed among groups (p > 0.05). After 45 days, for this variable, values reached ∼63%, 38%, 45%, and 28% for BG, BG/Col, BG/Mg, and BG/Col/Mg, respectively (Figure 3(a)). Statistical difference was observed between group BG and BG/Col/Mg at this last time point (p = 0.0187).

The values for ObS/BS, after 15 days, ranged between 3.8% and 5.2% for the studied groups with no statistical difference (p > 0.05). At day 45, the ObS/BS values were between 1.9% and 4.3% and, still, no statistical difference was observed among groups (p > 0.05; Figure 3(b)).

Immunohistochemistry

After 15 days of implantation, runx-2 immunostaining was noticed for all groups, mainly at the borders of the defect, granulation tissue, and neoformed bone (Figure 4(a)–(d)). The immunolabeling was also detected in the osteoblasts and medullary tissue. On day 45, runx-2 labeling was still found in the newly formed bone and remaining granulation tissue among the particles of the biomaterial, and, in some samples, in the medullary tissue (Figure 4(e)–(h)).

Likewise, at days 15 and 45 post-implantation, Rank-L immunoexpression was detected throughout the defect for all groups (Figure 5). Rank-L factor was found more evidently in the neoformed bone and in samples whose medullary and granulation tissues were still detected.

Immunohistochemistry: Semi-quantitative analysis

The findings for runx-2 and Rank-L semi-quantitative analysis are shown in Figures 6 and 7, respectively. The groups presented immunoexpression values ranging from 2.2 to 3.3 which are indicative of moderate immunostaining. Statistical difference was found for runx-2 immunolabeling for BG/Mg (∼3.2) compared to BG (∼2.2) at day 45 (p = 0.0345; Figure 6). No statistical difference was found for Rank-L (p > 0.05; Figure 7).

Mechanical test

After 15 days of implantation, the mechanical test indicated values ranging from 0.054 to 0.066 kN, 0.023 to 0.031 J, and 0.025 to 0.043 J for maximal load (kN), resilience (J) and tenacity (J), respectively. Over time, at day 45, increased values were found for the groups compared to the previous period, with values ranging from 0.080 to 0.096 kN, 0.033 to 0.038 J, and 0.038 to 0.044 J for maximal load, resilience, and tenacity, respectively. No statistical difference was noticed among groups (p > 0.05; Table 3).

Table 3.

Means ± SD for the mechanical evaluation of the tibiae.

	BG	BG/Col	BG/Mg	BG/Col/Mg
15 days
Maximal load (kN)	0.065 ± 0.026	0.061 ± 0.008	0.066 ± 0.008	0.054 ± 0.007
Resilience (J)	0.031 ± 0.013	0.025 ± 0.008	0.023 ± 0.003	0.023 ± 0.006
Tenacity (J)	0.043 ± 0.015	0.031 ± 0.014	0.025 ± 0.003	0.038 ± 0.016
45 days
Maximal load (kN)	0.085 ± 0.011	0.084 ± 0.011	0.080 ± 0.029	0.096 ± 0.013
Resilience (J)	0.034 ± 0.016	0.033 ± 0.007	0.038 ± 0.024	0.035 ± 0.008
Tenacity (J)	0.043 ± 0.018	0.038 ± 0.007	0.044 ± 0.030	0.042 ± 0.019

BG: bioglass; Col: collagen.

No statistical difference was found among groups (p > 0.05).

Discussion

This study aimed to investigate the in vivo performance of BG/Col/Mg composites on bone repair using tibial defects in rats. For this purpose, histopathological and immunohistochemical analyses, and mechanical test were performed in the harvested samples after 15 and 45 days of biomaterial implantation. Histopathological analysis showed that the materials degraded over time, leading to a progressive bone formation from the periphery to the center of the defect. Immunohistochemistry indicated that all groups presented runx-2 and Rank-L immunostaining, with increased runx-2 labeling for BG/Mg compared to BG at day 45. Mechanical tests of the tibiae indicated no statistical difference among groups.

All tested composites showed to be biocompatible, attracting osteoblasts and allowing newly bone formation in the defect area. Curiously, BV/TV and ObS/BS percentages of BG enriched with Col and Mg were not significantly increased compared to plain BG. These were not expected findings and do not corroborate, for instance, (i) with a previous study that demonstrated Col-mesoporous BG nanofibers promoted increased cell attraction and proliferation in rat calvarial defect model,⁴⁰ (ii) with Nijsure et al. who demonstrated that BG/Col/copper scaffolds improved osteoblast growth and attachment,⁴¹ (iii) with Han et al. and Huan et al. who studied Mg/Ca alloys and BG/Mg-based materials, respectively, showing that these materials stimulated osteoblastic cells proliferation.^26,27 In view of the present histopathological and morphometrical findings, however the chosen amount of Mg (∼3 wt%) was based on promising previous work of our group,²⁸ in the present in vivo scenario this amount could not be effective in order to promote any extra positive effect on bone formation. Hence, long-term additional animal studies, using higher quantity of this element (3 wt% < Mg < 5 wt%) may be required to verify and confirm the osteogenic potential of these new Mg-based composites.

Immunohistochemistry analysis showed runx-2 and Rank-L immunostaining for all samples. Runx-2 and Rank-L are key factors for mesenchymal progenitors differentiation into osteoblast, and resorption and remodeling of bone tissue by osteoclasts, respectively.^42–46 All treatments were capable of stimulating runx-2 and Rank-L immunostaining. More evidently, runx-2 expression was increased for BG/Mg compared to BG after 45 days. Several authors noticed an upregulation of runx-2 immunostaining in the presence of biomaterials or after other therapeutic interventions.^22,47–51 More specifically, Galli et al. investigated local release of Mg from mesoporous TiO₂ coating in tibia of rabbits and also observed a more pronounced runx-2 expression in the test specimens.⁵² Furthermore, Dai et al., studying calcium/magnesium-doped silica-based scaffolds incorporated with rhBMP-2, showed that this system promoted mesenchymal stromal/stem cells differentiation, as evidenced by the increased expression of runx-2.⁵³ Possibly, it may be suggested that the release of Mg ions at the implant sites stimulates an osteogenic microenvironment suitable for cell growth and proliferation. On the other hand, an unpredicted lack of difference was found among groups for Rank-L and this fact was also observed previously with similar composites but associated with low level laser therapy.²⁸ Still, all the composites were able of stimulating runx-2 and Rank-L immunostaining, as detected in both qualitative and quantitative assessments, which showed moderate immunostaining, positively influencing the osteoblast activity.

The mechanical evaluation of the tibiae showed no statistical difference for maximal load, resilence, and tenacity when comparing groups with and without Mg. Curiously, Zhang et al. did not find any significant difference while testing different Mg-Zn-Ca alloy scaffolds, at different periods, for in vivo repair either.⁵⁴ In contrast to that, earlier work by Staiger et al. stated the superior properties of magnesium in terms of biodegradability and mechanics, making this biomaterial promising for orthopedic applications.⁵⁵ Following this line, investigations by Khandaker and Tarantini showed high interface strength for bone-MgO particles.⁵⁶ As above-mentioned, however the selected quantity of Mg was based on an earlier work of our research group,²⁸ most probably this Mg amount was not sufficient to improve significatively the mechanical properties of the tibia. Still, in order to ilustrate the good mechanical properties of these Mg-based materials, though BG have presented significantly higher BV/TV compared to BG/Col/Mg at day 45, as well as being also expected the first to have improved mechanical properties compared to the latter, BG did not present enhanced values for biomechanics (0.085 kN) compared to BG/Col/Mg (0.096 kN). Thus, it is suggested that Mg content could counterbalance the expected better mechanics of BG compared to BG/Col/Mg, providing good mechanical properties to the last one.⁵⁵

Succinctly, our data on new BG/Col/Mg-based composites are quite promising and lead us to additional molecular and cell culture studies, and in vivo researches to clarify their osteogenic performance for bone tissue biomedical and orthopedics applications.

Conclusions

Biocompatible BG/Col/Mg composites were obtained, which led to a bone formation in tibial defects of rats. All materials presented osteogenic immunofactor expressions, with increased runx-2 labeling for BG/Mg compared to BG 45 days post-implantation and good mechanical properties. Further studies should be completed to validate the effect of BG/Col/Mg on bone repair, utilizing different animal models and amounts of Mg, as well as compromised and long-term situations, and critical bone defects conditions.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: PRGA thanks Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the scholarship (grants no. 2015/20704-8 and 2018/16328-9).

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