Collagen-chitosan-hydroxyapatite composite scaffolds for bone repair in ovariectomized rats.

Lesions with bone loss may require autologous grafts, which are considered the gold standard; however, natural or synthetic biomaterials are alternatives that can be used in clinical situations that require support for bone neoformation. Collagen and hydroxyapatite have been used for bone repair based on the concept of biomimetics, which can be combined with chitosan, forming a scaffold for cell adhesion and growth. However, osteoporosis caused by gonadal hormone deficiency can thus compromise the expected results of the osseointegration of scaffolds. The aim of this study was to investigate the osteoregenerative capacity of collagen (Co)/chitosan (Ch)/hydroxyapatite (Ha) scaffolds in rats with hormone deficiency caused by experimental bilateral ovariectomy. Forty-two rats were divided into non-ovariectomized (NO) and ovariectomized (O) groups, divided into three subgroups: control (empty defect) and two subgroups receiving collagen/chitosan/hydroxyapatite scaffolds prepared using different methods of hydroxyapatite incorporation, in situ (CoChHa1) and ex situ (CoChHa2). The defect areas were submitted to macroscopic, radiological, and histomorphometric analysis. No inflammatory processes were found in the tibial defect area that would indicate immune rejection of the scaffolds, thus confirming the biocompatibility of the biomaterials. Bone formation starting from the margins of the bone defect were observed in all rats, with a greater volume in the NO groups, particularly the group receiving CoChHa2. Less bone formation was found in the O subgroups when compared to the NO. In conclusion, collagen/chitosan/hydroxyapatite scaffolds stimulate bone growth in vivo but abnormal conditions of bone fragility caused by gonadal hormone deficiency may have delayed the bone repair process.

Use of collagen and auricular cartilage in bioengineering: scaffolds for tissue regeneration.

The aim of this study was the development of collagen and collagen/auricular cartilage scaffolds for application in dermal regeneration. Collagen was obtained from bovine tendon by a 72 h-long treatment, while bovine auricular cartilage was treated for 24 h and divided into two parts, external (perichondrium, E) and internal (elastic cartilage, I). The scaffolds were prepared by mixing collagen (C) with the internal part (CI) or the external part (CE) in a 3:1 ratio. Differential scanning calorimetry, scanning electron microscopy (SEM) analysis, microcomputed tomography imaging (micro-CT) and swelling degree were used to characterize the scaffolds. Cytotoxicity, cell adhesion, and cell proliferation assays were performed using the cell line NIH/3T3. All samples presented a similar denaturation temperature (Td) around 48 °C, while CE presented a second Td at 51.2 °C. SEM micrographs showed superficial pores in all scaffolds and micro-CT exhibited interconnected pore spaces with porosity above 60% (sizes between 47 and 149 µm). The order of swelling was CE < CI < C and the scaffolds did not present cytotoxicity, showing attachment rates above 75%-all samples showed a similar pattern of proliferation until 168 h, whereas CI tended to decrease after this time. The scaffolds were easily obtained, biocompatible and had adequate morphology for cell growth. All samples showed high adhesion, whereas collagen-only and collagen/external part scaffolds presented a better cell proliferation rate and would be indicated for possible use in dermal regeneration.

PVA/anionic collagen membranes as drug carriers of ciprofloxacin hydrochloride with sustained antibacterial activity and potential use in the treatment of ulcerative keratitis.

Devices such as contact lenses and collagen shields have been used to improve the antibiotic bioavailability of eye drops formulations in the treatment of ulcerative keratitis. Nevertheless, these devices are not sustained drug delivery systems, and a combination with eye drops is necessary. In animal patients, it requires constant supervision by trained personnel to avoid device loss, which increases the cost of treatment. In this study, PVA/anionic collagen membranes containing ciprofloxacin or tobramycin were prepared using two different methodologies, and the release, physical and antimicrobial properties were evaluated. The membrane containing ciprofloxacin was selected as a sustained drug delivery system with antibacterial activity against Staphylococcus aureus and Escherichia coli during 48 h. Despite to be opaque, due to its heterogeneous morphology, this membrane had the adequate mechanical strength, water content, hydrophilicity, water vapor permeability, and surface pH to interact with cornea without causing discomfort. In the surface of this membrane it was observed dispersed collagen fibrils which could serve as a substrate for corneal proteinases, contributing to the reduction in stromal damage and enhancing the epithelium regeneration. These results encourage the idea these membranes are new cost-effective and safe alternatives to treat corneal ulcers in animal patients.

Effects of the combination of low-level laser therapy and anionic polymer membranes on bone repair.

In view of the limitations of bone reconstruction surgeries using autologous grafts as a gold standard, tissue engineering is emerging as an alternative, which permits the fabrication and improvement of scaffolds to stimulate osteogenesis and angiogenesis, processes that are essential for bone repair. Polymers are used to mimic the extracellular bone matrix and support cell growth. In addition, bone neoformation can be induced by external factors such as laser irradiation, which stimulates bone metabolism. The objective of this study was to evaluate the regeneration of bone defects using collagen and elastin membranes derived from intestinal serosa and bovine auricular cartilage combined with low-level laser application. Thirty-six Wistar rats were operated to create a 3-mm defect in the distal metaphysis of the left femur and divided into six groups: G1 (control, no treatment); G2 (laser); G3 (elastin graft), G4 (elastin+laser); G5 (collagen graft); G6 (collagen+laser). The animals were sacrificed 6 weeks after surgery and the femurs were removed for analysis of bone repair. Macroscopic and radiological results showed the absence of an infectious process in the surgical area. This was confirmed by histological analysis, which revealed no inflammatory infiltrate. Histomorphometry showed that the formation of new bone started from the margins of the bone defect and its volume was greater in elastin+laser and collagen+laser. We conclude that newly formed bone in the graft area was higher in the groups that received the biomaterials and laser. The collagen and elastin matrices showed biocompatibility.

Suitability of the use of an elastin matrix combined with bone morphogenetic protein for the repair of cranial defects.

The use of biomaterials in medical and dental areas has become increasingly important due to the need to restore areas with bone loss or defects. This study analyzed the use of a new elastin polymer matrix combined with Bone Morphogenetic Protein for the repair of cranial defects in rats. Thirty rats were divided into five groups: control (C) defect without graft, E24 (defect filled with elastin matrix submitted to alkaline hydrolysis at 50°C for 24 h), E24/BMP (defect filled with elastin matrix treated at 50°C for 24 h plus BMP), E96 (defect filled with elastin matrix treated at 37°C for 96 h) and E96/BMP (defect filled with elastin matrix treated at 37°C for 96 h plus BMP). The animals were killed after 6 weeks. In the histological and microtomographic analysis, all groups showed bone growth from the defect margins remaining in this region without a marked inflammatory process, but in the E96/BMP group the lamellae were thicker and the collagen fibers more organized. Histometrically, the same group presented higher percentage of new formation (43.25 ± 3.72) in relation to the other groups. It was concluded that the support and delivery system formed by the elastin matrix associated with BMPs had a positive effect on the bone repair process.

Biomimetic mineralization of charged collagen matrices: in vitro and in vivo study.

Polyanionic collagen matrix prepared by hydrolysis side chain amides of asparagine and glutamine was mineralized in vivo, without inflammatory response, biodegradation, or resorption, with calcium phosphate deposited in close resemblance to the D-periodicity of collagen fibrils assembly. In vitro results with the same material produced mineralized collagen fibers with a similar morphology and chemical characteristics, suggesting that amide hydrolysis may have introduced into this matrix, signs for the controlled mineralization of collagen fiber. TEM indicated that amide hydrolysis occurred near the OVERLAP and GAP zones, as suggested by the significant reduction in inter-band distances in these regions. The lack of an inflammatory response associated to the similar mineralization pattern observed in vivo and in vitro suggests not only the biomimetic behavior of polyanionic collagen matrix, but also its potential uses as scaffold for bone tissue reconstruction. Based on these results, a model for the in vitro mineralization was also proposed.