A Composite Chitosan-Reinforced Scaffold Fails to Provide Osteochondral Regeneration

Several biomaterials have recently been developed to address the challenge of osteochondral regeneration. Among these, chitosan holds promises both for cartilage and bone healing. The aim of this in vivo study was to evaluate the regeneration potential of a novel hybrid magnesium-doped hydroxyapatite (MgHA), collagen, chitosan-based scaffold, which was tested in a sheep model to ascertain its osteochondral regenerative potential, and in a rabbit model to further evaluate its ability to regenerate bone tissue. Macroscopic, microtomography, histology, histomorphometry, and immunohistochemical analysis were performed. In the sheep model, all analyses did not show significant differences compared to untreated defects (p > 0.05), with no evidence of cartilage and subchondral bone regeneration. In the rabbit model, this bone scaffold provided less ability to enhance tissue healing compared with a commercial bone scaffold. Moreover, persistence of scaffold material and absence of integration with connective tissue around the scaffolds were observed. These results raised some concerns about the osteochondral use of this chitosan composite scaffold, especially for the bone layer. Further studies are needed to explore the best formulation of chitosan-reinforced composites for osteochondral treatment.


Imaging Acquisition
For the rabbits' explants, each sample was rotated by 180° with a rotation step of 0.4° and an average frame of 2. The nominal resolution was 9 μm. For the sheep explants, each sample was rotated by 180° with a rotation step of 0.4° and an average frame of 3. The nominal resolution was 17.5 μm. The acquired images were later reconstructed by the software NRecon (1.6.8.0) with corrections for alignment beam hardening and ring artefact reduction (Fig. 5).
The microtomographic sections were used to create 3D models of the analyzed object that allowed visualizations of the samples in space. Two specific volumes of interest (VOIs) were defined in each sample. TV1: Cylindrical VOI with diameter of 6 mm and height of 8 mm or diameter of 7 mm and height 5 mm for rabbits and sheep, respectively (used to evaluate the material and the new bone formation into the defect); TV2: VOI of the trabecular bone of the condyle in the area around the defect with the same height of TV1 (used to evaluate peri-implant bone).

Evaluation of Oxytetracycline Incorporation:
To assess bone tissue growth through dynamic histomorphometry, the animals received an i.m.
injection of oxytetracycline (30 mg/kg) (Terramicina 100, Pfizer Italy, Italy) before the end of the experimental time (two days on, ten days off, two days on).
Oxytetracycline incorporation was evaluated into 5 regions of interest (ROI) near or inside the scaffolds, rating mineral apposition rate (MAR) and bone formation rate (BFR) as previously described The 5 ROI were grabbed at 20x magnification using a light/fluorescence microscope connected to a digital camera and to an image analysis software (BX51, Olympus Optical Co. Europe GmbH, Germany). Oxytetracycline labelling was evaluated by applying the nomenclature and methodology of the American Society of Bone and Mineral Research (ASBMR) as follows:

1.
Mineral Apposition Rate (MAR, μm/day): The distance between the midpoints of two consecutive deposited and epifluorescent fronts of fluorochrome divided by the time between the labelling period.

2.
Bone Formation Rate (BFR/B.Pm μm2/μm/day): Obtained by multiplying the MAR value by the sum of half the single label perimeter (sL.Pm) and double the label (dL.Pm) perimeter.

Immunohistochemical Analysis: Details
After fixation, sections were extensively rinsed in PBS and permeabilized by an incubation in 0.3% hydrogen peroxide in PBS solution. Sections to be immunostained were pre-treated for antigen unmasking with 0.2% Pronase (Sigma, Mo, USA) solution in PBS. Subsequently, 10% normal serum was added to block nonspecific antibody binding, and the primary antibodies (Thermo Fisher Scientific Inc, USA) were applied. After rinsing in PBS, slides were incubated with appropriate biotinylated secondary antibody and with horseradish peroxidase-streptavidin complex (Bethyl Laboratories, Inc, TX). Sample reaction was developed with 3,3-diaminobenzidine substrate and permanently mounted. Figure 6: Results of calculated 3D parameters relevant to newly formed bone inside the defect and peri-implant bone inside TV2, on control (group 1) and on experimental group (group 2). Figure 7: Results of calculated 3D BV/TV, TbTh, TbN, and TbSp parameters relevant to newly formed bone inside the defect and peri-implant bone inside TV2, on control (group 1) and on experimental group (group 2).