Effect of calcium carbonate nanoparticles, silver nanoparticles and advanced platelet-rich fibrin for enhancing bone healing in a rabbit model

This study aimed to evaluate the efficacy of calcium carbonate nanoparticles (CCNPs) to induce new bone formation in a critical size segmental bone defect in rabbit’s radius when used alone, combined with silver nanoparticles (AgNPs) as a paste, or as a composite containing CCNPs, AgNPs, and advanced platelet-rich fibrin (A-PRF). Thirty-six adult apparently healthy male New Zealand White rabbits aging from 5 to 6 months and weighting 3.5 ± 0.5 kg were used. The animals were divided into four groups; control group, CCNPs group, CCNPs/AgNPs paste group, and CCNPs/AgNPs/A-PRF composite group. The animals were investigated at 4, 8, and 12 weeks post-implantation in which the healing was evaluated using computed tomographic (CT) and histopathological evaluation. The results revealed that CCNPs/AgNPs paste and CCNPs/AgNPs/A-PRF composite has a superior effect regarding the amount and the quality of the newly formed bone compared to the control and the CCNPs alone. In conclusion, addition of AgNPs and/or A-PRF to CCNPs has reduced its resorption rate and improved its osteogenic and osteoinductive properties.


Materials and methods
Animal model and housing.Animals were managed according to the guide for care and use of laboratory animals approved by the Animal Care and Use Committee, University of Sadat City-faculty of Veterinary Medicine, Sadat City, Egypt (registration number: VUSC-031-1-21) and was carried out under relevant guidelines and regulations.This work was done in compliance with the ARRIVE guidelines and regulations (https:// arriv eguid elines.org).
All animals have been accommodated and cared for according to the Egyptian animal welfare law (No.53,  1966).
Thirty-six apparently healthy adult male New Zealand White rabbits were used in this study.the mean age of the animals was 6 ± 1 months and its weights ranging from 3 to 4 kg.The animals were housed singly in stainless steel cages and were kept in humidity 60% with 12 h light/dark cycle and a temperature of 23 (± 2)°C.They had free access to standard diet and water during the whole period of the experiment.All rabbits were vaccinated against Rabbit Hemorrhagic Disease virus (RHDV) using (0.5 ml s/c, bivalent RHDV gel vaccine®, SERVAC Co.; A.R.E) and protected against external and internal parasites using (Dectomax®, Zeotis Co.; A.R.E).The experimented rabbits were randomly and equally divided into four groups (n = 9), each group equally divided into three subgroup according to the observation period 4th, 8th and 12th week (n = 3).

Bone substitutes preparation. CCNPs and AgNPs were synthesized in the Nanomaterials Synthesis and
Research unit, Animal Health Research Institute (AHRI), Agriculture Research Centre (ARC), Egypt.
Following the instructions of Hussein et al. 21in preparing CCNPs and Amer et al. 22 for preparing AgNPs.
Characterization of the prepared nanoparticles.
1. Dynamic Light Scattering (DLS) DLS showed that the average particle size of CCNPs was 88 nm while the average particle size of AgNPs was 10.4 nm.

X-ray diffraction (XRD)
The phase identification and structure of the CCNPs in which the presence of CCNPs can be observed at 20 of 23.0, 29.5, 36.0°.
A-PRF membrane was prepared just before the implantation according to Ghanaati et al. 18 .It has been prepared by obtaining 5 ml of whole blood from the central ear vein.The blood was centrifuged at 1500 rpm for 14 min.The formed PRF clot was dissected from RBCs and was compressed between two sterilized gauzes.CCNPs/AgNPs paste CCNPs/AgNPs/A-PRF composite were prepared on the same basis as described by Abd Elkawi et al. 23 .

Surgical procedure.
The mid-shaft of the right radius was exposed via 5 cm cranial skin incision at the fore arm.After the muscles were dissected, a 14 mm segmental defect was made using an electric saw.The defect was left empty in the control group (group A), filled with CCNPs paste in group (B), in group (C), the defect was reconstructed with CCNPs/AgNPs paste, while in in group (D), it was reconstructed with CCNPs/AgNPs/A-PRF composite.multidetector at the 4th, 8th, and 12th week post-operatively using a middle-frequency kernel, 0.9-mm-thick axial images.The obtained images were undergone three-dimensional (3D) multiplanar reconstruction using the RadiAnt DICOM image processing software (Medixant Co, Poznan, Poland).The quantitative bone densities at the operation site (the area of interest, AOI) and the adjacent bone (BAD) were also measured and correlated.
Histo-pathological evaluation.At each time point 4, 8, and 12 weeks, thirty-six rabbit were euthanized following the guideline of the American Veterinary Medical Association (AVMA), Euthanasia was done by injecting the rabbit with xylazine Hcl at dose 5 mg/Kg for sedation followed by thiopental sodium injection at dose 100 mg/Kg intravenously.Two cm bone specimens were harvested and fixed immediately in 10% buffered formalin for 72 h.The samples were decalcified using 10% EDTA di sodium for one month.Sections of 5 μm thickness were obtained, stained with Hematoxylin & Eosin stain, and covered with coverslips.The defects were evaluated by two pathologists using the Emery scoring system 24 (Table 1).

Statistical analysis.
The obtained values were reported as median, and standard deviations.A one-way ANOVA was used to carry out statistical analysis p ≤ 0.05 was considered statistically significant.All obtained values were analyzed using the SPSS software (version 20.0; IBM, America).

Results
Concerning the 3D images and the axial plane cross sections, the control group showed an empty defect at 4-and 8-weeks post-implantation with a few amounts of new bone in-growth without the appearance of any signs of remodeling (canalization) at the end of the study.On the other hand, the other groups showed new bone ingrowth at 4-and 8-weeks post-implantation with various degrees of defect filling and bone remodeling.At the end of the study group C and group D showed complete defect filling with new bone formation but only group D showed complete canalization (Fig. 1).
The results of the quantitative measurements of bone densities showed significant deference in normal radial densities in relation to different time points throughout the study (P = 0.000-0.031).While no significant difference was reported among the four experimental groups at the same observation period (P = 0.766-0.937).There was a significant difference in AOI density between all groups (P = 0.000) at all time points (P = 0.000).The AOI, group D showed relatively low density compared to group B and C. At the end of the observation period, the AOI density of group D was (1386 HU) which is very close to the normal bone density (1444.3HU) followed by group C (1501 HU).The control group showed the least AOI density (452.5 HU) among all tested groups and was found in the 4th post-operative week.Concerning BAD density, there was a significant difference in BAD density between all groups (P = 0.001-0.004)throughout all time points (P = 0.000) (Fig. 3).Group D had the least bone density between all examination groups.Which become (1434 HU) close the normal bone density by the end of the observation period (1444.3HU) followed by group C (Table 2) (Fig. 2).
Histological evaluation of the bone defect showed the superiority of group (D) over other groups.In group (D), the maturity of the newly formed bone started as early as by the end of the 4th week and become completely mature by the end of the 8th week post-implantation.On the other hand, bone maturation retarded till the end of the 8th, and 12th week post-implantation in group (C) and group (B) respectively.The control group failed to show ossification and rather formed fibrocartilage after 12 weeks (Table 3) (Fig. 3).
At four weeks observation period, histological examination showed cartilaginous transformation and appearance of the mature chondrocytes in most of the treated groups, the control group showed unorganized fibrous tissue filling the center of the defects with a large number of chondroblasts and osteoblasts at the bony edges.At eight weeks observation period, significant bone growth was seen in group (D) forming more dense compact bone, and group (C) showed large ossification centers expressed in osteonal canals with hypercellularity with osteoblasts, the groups (B) showed very few osteonal canal formation at the periphery of the defect.At twelve weeks observation period, group (D) and group (C) showed more condensation of the bone structure and calcium deposition which was higher in group (D), the groups (B) showed an increase of osteonal canals within the cartilaginous tissue with a high number of osteoblasts (Figs. 4, 5, 6, 7).

Empty gap 0
Filled with fibrous connective tissue only 1 More fibrous tissue than fibrocartilage 2 More fibrocartilage than fibrous tissue 3

Fibrocartilage only 4
More fibrocartilage than bone 5 More bone than fibrocartilage 6 Filled only with bone 7

Discussion
Management of large bone defects; particularly that resulted from infection, severe trauma, resection of tumors or cysts, or that resulted from the failure of treatment such as delayed or non-union presents a great challenge for veterinary orthopedists 25 .Different techniques have been established for the reconstruction of bone loss and overcoming these problems 26 .Modern techniques are based mainly upon the improvement of bone regenerative capability which can be achieved by using cells capable of bone formation when delivered alone or with an osteoconductive scaffold to a skeletal defect 27 , or by using biologically active materials to overcome the inherent limitations associated with the use of autografts, allografts, and xenografts 28 .A-PRF is widely used as a bone substitute.It's known for its good bone-regenerating capacity 16 .As a fibrin biomaterial A-PRF contains the favorable components present in blood samples such as a large number of platelets, cytokines, and leukocytes 29 .There are many advantages of using PRF (the second generation of concentrated platelets) instead of PRP (the first generation) in bone healing.Preparation PRF does not need an anticoagulant (which interferes with the healing process), also it is prepared by one-step centrifugation at room temperature www.nature.com/scientificreports/compared to PRP which needs two-steps centrifugation.Also, PRF gradually releases its growth factor content for a long period offering a continuous supply during the healing process, while PRP releases about 80% of its GFs content in the first 24 h 30 .
CCNPs is extensively used in drug delivery system.It is easily prepared, cost-effective, and had no toxic effect on animal models 31 .Although it has structure resembles the mineral structure of a normal bone, There is not enough data to discuss its role in accelerating bone healing 32 .

Table 2.
Results of quantitative bone densities of normal bone, new bone in growth (AOI density) and density of bone adjacent to the defect area (BAD density) by CT and expressed as mean ± SD.The data showed a significant difference in AOI and BAD densities between all groups at all time points (P = 0.000-0.004).*,**,***Medians and ranges with different asterisks superscripts in the same column are significantly different at P < 0.05.a,b,c Medians and ranges with different small superscripts letters in the same row are significantly different at P < 0.05.There is a significant difference between all groups (P = 0.000) throughout all time points (P = 0.000).At the end of the observation period the AOI density of group (D) (1386) was very close to the normal bone density (1444.3)followed by group (C) (1501).(C) Showing the BAD density of all examined groups at different time points (4,8, and 12 weeks).There is a significant difference between all groups (P = 0.001-0.004)throughout all time points (P = 0.000).As shown in the results group (D) had relatively least bone density and at the end of the observation period the BAD density (1434) was relatively the same as the normal bone density (1444.3).
AgNPs are well known for its antibacterial, antifungal, antiviral, anti-inflammatory, and good osteogenic activities 33,34 .It is also, available, easily prepared, and cost effective 35 .Its use in the field of bone regeneration was limited in invitro application with calcium carbonate to reduce the prevalence of infection 36 .To our knowledge, this may be the first report which evaluated and described the in-vivo bone regenerating capacity of CCNPs when used alone and in combination with AgNPs and A-PRF to fill critical size bone defects in a rabbit model.
In our previous work, the prepared CCNPs and AgNPs biomaterials have been evaluated using DLS, TEM, and XRD examination.Our results revealed that CCNPs appeared as agglomerated spheres with 88.4 nm average particle size, with a characteristic peak positioned 20-29.5 at a wavelength of 1.54060.While AgNPs appeared as ultrafine uniform, well-dispersed spherical nanoparticles with 10.4 nm average particle size 23 .These results matched well with the results of Hill et al. 37 , which confirmed the nano-scale size of the used nanomaterials in this study.
The age of the used experimental animals, might be an important factor affecting the behavior of the examined biomaterials.In the present study, 6-month-old rabbits were included.Although long bones have reached about 95% of their adult length in rabbits by 16 weeks of age 33 , the skeletal growth of New Zealand White Rabbits complete between 19 and 32 weeks.In view of this, the use of rabbits older than 6 months should be recommended for bone regenerative studies 38 .The implantation time also plays a role as well.Some reports showed nonbridging of rabbit radial defects at 4-8 weeks of implantation 39 .In this study the observation period extended to 12 weeks.Our radiographic and histopathologic results showed that this period was sufficient for complete healing of bone defect.These result was found in accordance with 33,38 The obtained results of the quantitative analysis of the normal bone densities at different observation periods revealed no significant difference among the four experimental groups at each time point, However, it showed a significant difference at different time points.These data agreed with 40,41 , the authors stated that age was weakly but positively associated with cortical density at the tibial diaphysis among the healthy rabbits in which bone mineral density increases during skeletal maturation and continues four to eight weeks after the closure of the growth plates.
BAD and AOI densities values could be indicative of the resorption rate and pattern of the grafted material, the amount, and the quality of the newly formed bone.By tracking BAD and AOI densities curves throughout the study period, it has been noticed that BAD and AOI densities levels showed a marked reduction by the end of the 4th week as a result of active bone reaction at the defect periphery accompanied by resorption of the grafted materials.Group (D) showed the least value compared to other groups.This in turn supports the hypothesis Table 3. Showing the histological score evaluation of the four experimental groups at 4, 8, 12 weeks post operation.The data showed a significant difference between all experimented groups at all time points (P = 0.000-0.001).*,**,***Medians and ranges with different asterisks superscripts in the same column are significantly different at P < 0.05.a,b,c,d Medians and ranges with different small superscripts letters in the same row are significantly different at P < 0.05.The histopathological score showed great significant differences in the histopathological score at 4,8 and 12 weeks of the observation period among the four experimental groups (P = 0.000).At the end of the observation period (12 weeks), group B couldn't make complete healing although the presence of some newly formed bone, while group C&D showed complete healing with score = 6-7 respectively at the end of the observation period with a statistical difference (*) between them versus the two other groups (group B and control).
that the addition of A-PRF to CCNPs/AgNPs paste greatly reduced its resorption rate.Throughout the rest of the observation period (8-12 weeks), both variables showed a gradual increase indicating active new bone formation to reach its maximum level by the end of the 8th week in all tested groups compared to the control.The same data was obtained by Ou, et al. 42 , the authors noticed that the density increased with healing duration in each group, which can be attributed to mineral deposition.After the end of the 8th week, both variables showed a gradual reduction in all groups compared to the control which indicates bone remodeling.At the end of the observation period, BAD and AOI densities were approximately equal to the normal bone density in group (C) and group (D) which indicate complete bone remodeling.
Our histological findings confirmed the former radiographic observation.It showed a marked increase of osteogenesis with new bone formation in groups (B), (C) and (D) compared to the control one which failed to show ossification and rather formed a fibrocartilage after 12 weeks.Osteogenesis and new bone formation in group (D) started by the end of the 4th week and became mature by the end of the 8th-week post-operation compared to other groups.This can be attributed to the improved bone cellular functions of the used nanomaterials compared to their micron-sized counterparts.As well as, the biological role of blood derivative (A-PRF) in the stimulation of neo-vascularization, and cellular migration.These data agreed with that reported by 37,43,44 whom found that using autologous PRF in the induced gap has benefits for organizing the formative cell (especially osteoblast), formation of neovascularization and more rapid and faster apposition of the bony matrix with its mineralization process without significant inflammatory response, necrosis, or foreign body reactions demonstrating good osteoconductive and biocompatibility of enriched graft.This better healing potential noticed could be attributed to the osteoinductive effect of growth factors 45,46 .
In the same context, the addition of AgNPs to CCNPs showed improved osteogenesis of the implanted graft in which the defect was filled by new compacted bone with signs of remodeling and calcium deposition at the end of the observation period.These data are a confirmation of the study carried out by Zhang et al. 47 and Kumar et al. 8 in which the authors reported that AgNPs' has proliferative and osteogenic differentiation induction effects on MSCs and promotion of fracture healing in the osteogenesis of mouse model.They stated that AgNPs could promote the formation of the fibrous joint and subsequently end joining of the fractured bone via multiple routes: chemo-attraction of MSCs and fibroblasts to migrate to the fracture site, induction of the proliferation of MSCs and fibroblasts, and induction of osteogenic differentiation of MSCs.
In conclusion, the present study revealed that CCNPs alone have limited efficacy in bone generation and the addition of A-PRF and/or AgNPs to CCNPs reduced its resorption rate compared to CCNPs alone as well as improved its osteogenic and osteoinductive properties by husting of new bone formation.

Figure 2 .
Figure 2. Showing (A) the normal density of the radius of the four experimental groups.It showed a marked increase with animal age with no significant difference among the experimental groups at each evaluation timepoint (P = 0.766-0.937).(B) the AOI density of all examined groups at different time points (4,8, and 12 weeks).There is a significant difference between all groups (P = 0.000) throughout all time points (P = 0.000).At the end of the observation period the AOI density of group (D) (1386) was very close to the normal bone density (1444.3)followed by group (C) (1501).(C) Showing the BAD density of all examined groups at different time points (4,8, and 12 weeks).There is a significant difference between all groups (P = 0.001-0.004)throughout all time points (P = 0.000).As shown in the results group (D) had relatively least bone density and at the end of the observation period the BAD density (1434) was relatively the same as the normal bone density (1444.3).

Figure 3 .
Figure3.The histopathological score showed great significant differences in the histopathological score at 4,8 and 12 weeks of the observation period among the four experimental groups (P = 0.000).At the end of the observation period (12 weeks), group B couldn't make complete healing although the presence of some newly formed bone, while group C&D showed complete healing with score = 6-7 respectively at the end of the observation period with a statistical difference (*) between them versus the two other groups (group B and control).

Figure 4 .
Figure 4. Microscopic appearance of the control group showed at 4 weeks post-operative (PO) (1) the defect filled with unorganized fibrous tissue (F) with a large number of chondroblasts and osteoblasts (black arrows) at the bony edges.At 8 weeks PO (2), the defect was filled with cartilaginous tissue formation (C) with mature chondrocytes within organized fibrous tissue (F) while at 12 weeks PO (3), the defect was filled with fibrocartilage (FC) characterized by mature chondrocytes (C) in separate lacunae within fibrous tissue bundles.

Figure 5 .
Figure 5. Microscopic appearance of CCNPs group (group B) showed at 4 weeks PO (1), the defect filled with the cartilaginous transition from fibrocartilage characterized by migration of a large number of chondrocytes (Co) in separate lacunae within the fibrous tissue bundles (F).At 8 weeks PO (2), the defect was filled with large hyaline cartilage (C) with few osteonal canals on the peripheries of the defect (O), while at 12 weeks PO (3), the defect showed an increase in osteonal canal (O) formation within the calcified cartilage with a high number of osteoblasts (OB) within the osteonal canals.

Figure 6 .
Figure 6.Microscopic appearance of CCNPs/AgNPs group (group C) showed at 4 weeks PO (1), the defect filled with cartilaginous transition phase with formation of hyaline cartilage (H) and multiple mature chondrocytes in lacunae.At 8 weeks PO (2), the defect showed formation of fibrous connective tissue and woven bone (W) migrating to replace hypertrophic chondrocytes (Co) at the cartilage bone junction, while at 12 weeks PO (3), the defect showed the formation of the haversian system from osteonal canals (O) and trabecular bone condensation into compact bone (B) with calcium deposition (Ca).

Figure 7 .
Figure 7. Microscopic appearance of CCNPs/AgNPs/A-PRF group (group D) showed at 4 weeks PO (1), the defect filled with multiple osteonal canals (O) formation with a large number of osteoblasts (OB) forming trabecular bone (T) with active calcium deposition.At 8 weeks PO (2), the defect showed multiple osteonal canals (O) with compact bone formation (B) and calcium precipitation (Ca), while at 12 weeks PO (3), the defect showed the formation of the haversian system (H) with ossification and dense compact bone formation (B) with calcium deposition (Ca).