Analysis of the scaffolds
Figure 1 shows the scaffolds and their respective morphologies obtained with the different methods of hydroxyapatite incorporation. The macro images showed no visual differences between the two scaffolds, both presented as three-dimensional structures of white color, similar size, and porous and homogeneous aspect. The porous structures observed in SEM images showed that hydroxyapatite (arrows) was present and distributed throughout the polymeric matrix in both cases, first indication that both adopted hydroxyapatite incorporation methods were successful.
Macroscopic images of the scaffolds (left) and surface scanning electron photomicrographs (SEM, right) at ×500 magnification. Hydroxyapatite (arrows) was distributed throughout the polymer matrix.
Bertolo et al. (2019) found that, although not significant, CoChHa1 scaffolds had pores with smaller diameters (17.7 ± 6.8 µm) than CoChHa2 scaffolds (21.1 ± 8.5 µm)6. Moreover, the distribution of these pores was more homogeneous for CoChHa2 (around 33.33% of the pores with 20–30 µm of diameter), indicating that the method of phosphate incorporation into the scaffolds had a great influence on their morphological properties, which may affect the regenerative behavior of the scaffolds in vivo6. The pores of the scaffolds used in this study were significantly smaller than those found by Rahman et al. (2019), who prepared scaffolds of rabbit collagen, shrimp chitosan and bovine hydroxyapatite for restoration of defected maxillofacial mandible bone and found pores ranging from 101.69 ± 17 µm to 273.43 ± 49 µm39.
Macroscopic and radiologic analysis of the bone defect
Macroscopic analysis of the defect area in all rats showed good healing of the soft tissues, as demonstrated by the absence of necrosis, cysts, fibromatosis, abscesses and any evidence of a subcutaneous or deeper tissue inflammatory process. In the bone area, there were also no signs of osteomyelitis, secondary fractures or pseudarthrosis that would suggest any infectious complications (Fig. 2). These good outcomes of the animals may be explained by the fact that the experimental protocol followed the ARRIVE guidelines and principles of the NC3Rs initiative. The animals were monitored for the expression of pain by observing whether the animal was apathetic, depressed, aggressive, or overexcited, such traits being variable in their usual behavior. Possible changes in gait, posture or facial expression were also observed, and appearance, water intake, feeding and clinical symptoms were investigated. There were no complications that needed to be reported and no diseases or signs recommending the removal of an animal (clinical outcome) were observed40. In addition, our research group has experience in the method used, as demonstrated by previously published studies11,41,42,43.
Macroscopic and radiologic images of the groups of non-ovariectomized and bilaterally ovariectomized rats. Note the absence of infections and bone rarefaction that would indicate an intense immune response to the scaffolds. (a) Macroscopic image of the defect area in the left tibia after induced painless death. (b) Macroscopic image of the dissected left tibia. (c) Radiographic image of the left tibia. The arrows indicate the bone defect area in the proximal metaphysis of the left tibia.
The radiographic images showed a round radiolucency of the defect area in the proximal tibial metaphysis and radiopacity of the cortical margins of the tibia, demonstrating the absence of deformities or any other type of change in the bone structure (Fig. 2). These results are compatible with studies on the tibial metaphysis, an experimental model widely used for the investigation of bone repair and biomaterial filling, especially in non-critical defects44,45. Thus, the two types of scaffolds used in this study showed biocompatibility in critical bone defects, confirming the biocompatibility of the materials used, both polymers and the inorganic phase incorporated on them19. Zugravu et al. (2012) also reported this advantage for collagen/chitosan/hydroxyapatite scaffolds in in vitro studies46. Furthermore, the findings demonstrated that gonadal hormone deficiency did not exacerbate the local inflammatory response.
Morphological analysis of the bone defect area
The formation of new bone in the defect experimentally induced in the proximal tibial metaphysis of rats was observed in all groups analyzed; however, its morphology differed between the control groups and the experimental groups treated with the biomaterial and submitted to ovariectomy. The new bone projected from the margins of the defect but exhibited peculiar histological characteristics that differed between the groups studied and from the original bone of each rat. New formed bone contained lacunae filled with osteocytes that were arranged in various directions. The medullary canal was preserved and filled with hematopoietic tissue and bone trabeculae. Resorption of the biomaterials differed between the grafted groups (Fig. 3).
Photomicrographs of histological slides stained with Masson’s trichrome at ×4 (a) and ×10 magnification (b) obtained from the groups of non-ovariectomized and bilaterally ovariectomized rats. The arrows point to areas of bone neoformation. The star indicates the presence of biomaterial undergoing bioresorption. Scale bar: 20 µm.
There is still a preference for the use of autologous grafts for bone grafting; however, due to the need for two surgical beds (donor and recipient area), morbidity and limited availability, advantages arise in the use of synthetic biomaterials for bone tissue regeneration47. Three-dimensional scaffolds are particularly interesting in tissue engineering since they can act as structures to accommodate cells and support tissue growth, providing support for cell adhesion, proliferation, and migration48. The creation of a bone defect triggers a sequence of events in the local microenvironment, including the migration of inflammatory and proliferative cells of bone tissue, compatible with the remodeling process. These events allow the bone to respond and to adapt to functional changes, as observed in the present study49. The resorption rate of biomaterials differs depending on the material used. For example, particulate dentin grafts are characterized by a high resorption rate after 24 months as well as by bone substitution without inflammation. Since dentin particles have open tubes, capillaries can access their interior, resulting in rapid resorption50.
There is currently increasing interest in composites consisting of natural polymers (like collagen and chitosan) and hydroxyapatite, which form biocompatible scaffolds with interconnected pores that have a satisfactory osteogenic potential39,51,52. Collagen type I is the main component of the extracellular matrix and bone tissue in humans, while hydroxyapatite is the second most abundant component in bone39, which can be prepared synthetically with specific nano-sized pores for adequate deposition in the 3D microarchitecture of the scaffolds, called nano-calcium phosphate6.
Collagen matrices containing nano-calcium phosphate have shown osteogenic potential in critical bone defects53. Recently, synthetic calcium phosphate has been reported to stimulate biomineralization in collagen-based bone substitutes54. There is also evidence that chitosan plays an important role in the strengthening of the microarchitecture of these polymer matrices, whose biodegradability can be adapted according to the proportion of chitosan or hydroxyapatite52,55.
Specifically, in non-ovariectomized rats (G1, G2 and G3), the formation of irregular and immature bone was observed in the control group (G1, NO-C), including joining of the margins of the bone defect that contained cavities and spaces but without the interposition of connective tissue. In G2 (NO-CoChHa1), remnants of the biomaterial surrounded almost entirely by newly formed bone were found; however, bone neoformation was not sufficient to fill the entire defect due to the presence of connective tissue in the defect area. In G3 (NO-CoChHa2), the defect closed due to the volume of new bone formed, in a more compact way and promoting the union of the lesion margins, without the presence of connective tissue. Also in G3, there were remnants of the biomaterial inside the medullary canal, which, in turn, was filled with hematopoietic tissue, and several bone trabeculae. This feature was only observed in this group (Fig. 3). These microscopic findings agree with studies on bone repair that report the gradual centripetal substitution of the implanted biomaterial with newly formed bone, demonstrating the biocompatibility of the scaffolds used in this experiment56,57,58.
Histological analysis indicated a superior osteogenic potential of CoChHa2, a scaffold in which calcium phosphate was incorporated ex situ into the mixture of collagen gel and chitosan powder. The SEM images of this scaffold had already revealed a porous morphology suitable for growth and cell proliferation. The more homogeneous pore distribution might have been a factor to improve the osteogenic potential of CoChHa2, which was also about 7% less porous and absorbed around 500% less phosphate buffer saline (PBS) than CoChHa1 scaffolds. Moreover, X-rays diffraction patterns of hydroxyapatite (2θ = 32°) were present in both diffractograms in the study of Bertolo et al. (2019)6; however, hydroxyapatite influence on bone formation might have been greater in CoChHa2, that presented more intense and better-defined peaks (i.e., greater crystallinity, less influenced by collagen and chitosan presence).
According to the histological results, CoChHa2 stimulated greater bone neoformation in the tibial defect area of the animals. Regarding CoChHa1, bone neoformation also occurred around the defect area, but to a lesser extent. In conclusion, the two scaffolds studied here can be indicated for bone repair, since they both present interconnected pores and three-dimensional structures with proven presence of hydroxyapatite; however, there are differences in the time and rate of bone formation, factors directly related to the morphological and physicochemical properties (porosity, absorption, degradation) of the materials, as well as with the availability of the calcium phosphate phase. Scaffolds with more homogeneous porous and with hydroxyapatite more available in the polymeric matrix tend to accelerate the osteogenic process, which is a good feature when working with short recovery periods.
In ovariectomized rats, the formation of thinner bone along the defect was observed in G4 (O-C); however, the space was not completely closed given the presence of connective tissue. In G5 (O-CoChHa1), the young bone was compact at the margins of the defect but more trabecular and bordering the lower surface of the biomaterial which, in turn, was not completely reabsorbed. In G6 (O-CoChHa2), the biomaterial remained intact, with little bone formation around it, and a predominance of connective tissue was thus observed (Fig. 3). Analysis of the scaffolds in ovariectomized rats treated with CoChHa1 (G5) and CoChHa2 (G6) showed less bone formation in the two groups when compared to the respective non-ovariectomized groups; nevertheless, both scaffolds exerted a satisfactory effect as demonstrated by the onset of bone repair, although the process was slower. Evaluating polymer scaffolds in the femur of ovariectomized rats, Cunha et al. (2008) concluded that bone formation is dependent on the properties of the biomaterials as well as on the quality of the host bone tissue59.
Preclinical studies using an experimental model similar to that employed in this study have attempted to improve the formation of new bone in rats submitted to ovariectomy (or other models of osteoporosis induction) using tissue engineering techniques. Zhang et al. (2022) developed a new class of copper-alloyed titanium (TiCu) alloys with excellent mechanical properties and biofunctionalization60. The authors induced osteoporosis in rats to study the effect of osseointegration and the underlying mechanism of TiCu. The alloy increased fixation stability, accelerated osseointegration, and thus reduced the risk of aseptic loosening during long-term implantation in the osteoporosis environment. The study of Zhang et al. (2022) was the first to report the role and mechanism of a copper-alloyed metal in promoting osseointegration in the osteoporosis environment60.
Current technologies permit to connect different active functional groups by modifying their configuration or surface. These changes can significantly broaden the range of applications and efficacy of chitosan polymers61. Chitosan, calcium phosphate and collagen and their combination in composite materials meet the required properties (biocompatible, bioactive, biodegradable, and multifunctional) and can promote biostimulation for tissue regeneration52. In some situations of alteration by pathologies, the repair process and healing rate may be compromised even when these biomaterial are used62,63.
Staining with Picrosirius red under polarized light showed collagen birefringence in the extracellular matrix of tissue present in the defect area of all groups (Fig. 4). Picrosirius red is a dye that selectively stains connective tissue, enabling the qualitative analysis of collagen fibers. When observed under polarized light, this stain permits to differentiate especially type I and type III fibers based on the difference in interference colors, intensity and birefringence of the stained tissues64. New bone formation was characterized by red–orange birefringence, corresponding to the osteoid matrix, which gradually changed to yellow-green. Similar findings have been reported by Della Colletta et al. (2021)65.
Photomicrographs of histological slides stained with Picrosirius red obtained from the groups of non-ovariectomized and bilaterally ovariectomized rats and examined under an optical microscope (a) and under polarized light (b) at ×10 magnification. The black arrows point to areas of bone neoformation. The yellow arrows indicate the presence of collagen fibers in the extracellular matrix. The star indicates the area occupied by remnant biomaterial after bioresorption. Scale bar: 20 µm.
Histomorphometric and statistical analysis of newly formed bone volume in the defect area
The following means and standard deviations of the percent volume of newly formed bone in the tibial defect were obtained for the groups studied: 69.20 ± 3.3 (G1); 67.08 ± 4.5 (G2); 92.12 ± 0.5 (G3); 44.63 ± 5.3 (G4); 43.42 ± 9.9 (G5), and 44.59 ± 5.2 (G6). Statistical analysis revealed no significant difference between the ovariectomized groups (G4 vs. G5, G4 vs. G6, G5 vs. G6), while comparison of the non-ovariectomized groups showed a higher percent volume in G3 (NO-CoChHa2). Comparison of non-ovariectomized animals with their respective ovariectomized counterparts (G1 vs. G4, G2 vs. G5, G3 vs. G6) revealed a higher bone volume in the non-ovariectomized groups.
These results demonstrate that the biomaterials were unable to contribute to bone volume gain when a hormonal component resulting from experimental ovariectomy is involved. On the other hand, the bone volume increase was significant in non-ovariectomized animals grafted with the CoChHa2 scaffold (collagen/chitosan/hydroxyapatite, method 2). The success of biomaterials for fracture fixation in osteoporotic patients, or simply for bone augmentation, is compromised by poor bone quality and decreased osteoblastic activity. Further research, driven by clinical demand, is therefore needed to address this issue. The growing elderly population and the different associated pathologies require studies that involve close cooperation between basic research, applied research, clinical research, and regulatory bodies66.