In Vivo Studies
Titanium alloys and cobalt chrome have outstanding resistance and biocompatibility with high specific strength. So they are widely applied in dental, orthopaedic and other implants. Titanium fiber greatly improved the rate of repair bone defects. Hirota et al. applied a molecular precursor method, which is a thin hydroxyapatite (HA) film can be opportunely coated onto the titanium fiber web without damaging the interior structure (Fig. 7.9) . The in vivo implantation made in rat cranial bone defects shows that the quantity of newly formed bone is greatly higher in the HA-coated titanium fiber mesh than in the uncoated scaffold. 2 weeks later, the corresponding new bone formation ratio is 35.8% and 7.1%, respectively while the figures increase to 45.1 and 56.2%, demonstrating the earlier osteoinduction of thin HA coatings, which is interrelated to capability of HA to strengthen the formation of apatite crystals brought by human osteoblasts during the culturing process.
Water-soluble phosphorylated chitosan (P-chitosan) fiber is known to increase osteoblastic activity and disodium (1-4)-2-deoxy-2-sulfoamino-p-D- glucopyran- uronan(S-chitosan) fiber can inhibit the activities of giant cell tumor of bone [123, 124]. Wang et al. developed a novel bone repair materials incorporating P- and S-chitosan fiber, and tested osteointegration properties of the materials with a standard ulna bone defect model . They prepared composite scaffolds: S-chitosan fiber and P-chitosan fiber mixed separately with PLGA/tricalcium phosphate (TCP) to 1,4-dioxanesolution. A rapid prototyping machine was used to fabricate PLGA/ TCP/P-chitosan (P) and PLGA/TCP/S-chitosan (S) scaffolds. Similarly, Chen et al. further studied on the biocompatibility of scaffolds materials, and they found that new bone formation and mineralization took place within the pores of the scaffold and then remodeled together with degradation of the scaffold . There is a significant enhancement of bone regeneration in the groups of P-chitosan fiber and PSP-chitosan fiber compared to the control group at 12 weeks post-operation.
Fig. 7.9 Scanning electron microscope of hydroxyapatite (HA)-coated titanium fiber without damaging the interior structure: (a) Stereoscope finding of apatite crystal after 14 days of human osteoblast culture. Apatite crystals are deposited on titanium fiber (arrows). Scale bar 200 pm. (b) human osteoblast cultured in HA-coated titanium fiber web for 7 days. Human osteoblast extends projections to adhere to titanium fibers, secreting calcification matrix (arrows). Scale bar 20 pm (Adapted with permission from Ref. . Copyright 2012 Elsevier Ltd)
In addition, Li et al. prepared nano-hydroxyapatite/collagen/PLA (nHACP) scaffolds reinforced by chitin fibers to repair the goat shank bone defect. They found that the fiber reinforced scaffolds could successfully repair the 40-mm adult goat shank bone defect while the bare nHACP implants could not . They evaluated the performance of bone growth in the experimental groups during 15 weeks by radiograph, mechanical strength, histology and bone mineral density. The results demonstrated that chitin fibers reinforced nHACP implants showed satisfactory repairing defect capacity. Figure 7.10 showed the radiographs in the experimental groups during 15 weeks after surgery. For the defect group, we can see that there was hardly no new bone formation at 15 weeks after surgery. For the PLLA group, there was a little new bone formation at 15 weeks after surgery and the defect was far from the repair. For the the nHACP group, some new bone formed at the both ends of the defects at 15 weeks after surgery, which was significantly more than that for the PLLA group. However, the new bone have not been connected, showing that the defect was not fully repaired because the defect was too large for the nHACP/ CF implants. In contrast, for the fiber reinforced group, at only 5 weeks after surgery continuous callus appeared. Interestingly, marrow cavity in continuous new cortical bone could be observed at 10 weeks after surgery. At 15 weeks after surgery, the newly formed bone were successfully connected, and the bone defect was smoothly repaired. In addition, the mean breaking load of the reinforced group was higher than that in both pure PLLA and nHACP groups. Moreover, the mean breaking load of the implants for the reinforced group raised much quicker from 5 to 15 weeks than that for the nHACP group. At 15 weeks after surgery, the mean breaking load of the implants for the fiber reinforced group was about three times as that for PLLA group and about twice as that for nHACP group. Figure 7.11 showed the histological appearances of the defect tissue for all the experimental groups at the three time points. For the defect group, no osteoblasts but only fibrous-connective
Fig. 7.10 Radiographs in defect group at 15 weeks after surgery (a);PLLA group at 5 weeks (b1), at 10 weeks (b2) and 15 weeks (b3); nHACP group at 5 weeks (cl), at 10 weeks (c2) and at 15 weeks (c3); the fiber reinforced nHACP group at 5 weeks (dl), at 10 weeks (d2) and 15 weeks (d3), showing that the fiber reinforced group evidently enhanced the continuous cortex of regenerated bone and a marrow cavity at 10 weeks, and achieved full bone defect repair at 15 weeks (Adapted with permission from Ref. . Copyright 2006 Elsevier Ltd)
tissue can be found at 15 weeks after surgery. For the PLLA group, hardly any osteoblasts but a lot of mononuclear cells (white arrow area), neutrophilic granulocytes (red arrow area) could be observed, indicating that foreign-body feedbacks happened. Even at 15 weeks after surgery, no osseous callus could be found. For the the nHACP group, although not only osteoclasts (white arrow area) but also osteoblasts (red arrow area) were found at 5 weeks after surgery, and fibrous callus (red arrow area) could be observed at 10 weeks after surgery, no mature bone generated at 15 weeks after surgery. In contrast, for the fiber reinforced group, a lot of osseous callus (red arrow area) could be found at only 10 weeks after surgery. Most importantly, bone lacunas (red arrow area) and the bone cells (white arrow area) in the osseous callus could be observed at 15 weeks after surgery, indicating that mature new bone has generated. Furthermore, the analysis results of bone mineral density showed that the value hardly changed during the whole 15 weeks of implantation for the defect group, and although the bone mineral density for PLLA and nHACP increased with the increase of implantation, the value for the fiber reinforced group increased most. At 15 weeks after surgery, the mean bone mineral density for the fiber reinforced group was about twice as that for the nHACP group.
Fig. 7.11 (continued)
In 2016, Li et al. further provided a novel method to study the biocompatibility of nanoscale scaffolds reinforced by fibers or tubes for tissue engineering in vitro and in vivo . They used the in vitro biodegradation products of nanohydroxy- apatite/collagen (nHAC), nHACP, and the chitin fiber reinforced nHACP (nHACP/ CF) in the D-Hank’s Balanced Salt Solution as the testing solution, and found that. The results showed that the survival rate of the neutrophils was almost same in the different three testing solutions. But during the whole testing period the tumor necrosis factor-alpha and lactate dehydrogenase of the cells in the nHACP/CF testing solution were found lowest. In addition, in vivo experiments showed that hardly no inflammation reactions could be observed for the nHACP/CF group after the implantation into the subcutaneous dorsum of mice for 2 weeks after surgery while very serious inflammation reactions appeared for nHAC and poly (L-lactic acid) groups (Fig. 7.12). The results indicated that the addition of the reinforcing fibers effectively coordinated the interactions of the components, thereby appropriately controlling the local metal iron concentration and pH value at the implantation sites, which directly improved the biocompatibility of the scaffolds. Their study showed by a novel method that some appropriate fibers or tubes have the potential to not only enhance the mechanical properties of tissue engineering scaffolds but also improve their biocompatibility.
Moreover, Srivastava et al. inserted their composites in linking the big osteoperiosteal gap in rabbits, assessing the role of polyester resin composites strengthened by unidirectional carbon fiber, and filled by bone marrow impregnated tricalcium phosphate-polyvinyl alcohol with radiological, histological and clinical methods . In their study, 20 healthy mature Indian rabbits were operated independently and implanted with the same composites. After the implantation, incision wound was checked every day for 7 days to avoid the gaping, swelling and infection of wound even skin necrosis. The rabbits were observed for 12-32 weeks (Fig. 7.13). The single particle and new bone formation were showed in the histopathology of tricalcium phosphate (purple) neocapillarisation. They implanted polyester resin composites filled with tricalcium phosphate-polyvinyl alcohol and strengthened by carbon fiber into the bone marrow of rabbit to bridge large osteoperiosteal gap. The clinical observations results performs a normal body weight improving. The roentgen graphic and histological observations of implanted points showed that the new bones form on the distal and proximaljunction side. Tricalcium phosphate as a good bone conduction material allowed bone growth, because of which the proximal
Fig. 7.11 (continued) Histological appearances of the tissue in defects for defect group at 15 weeks after surgery, (a); PLLA group at 5 weeks (b1), at 10 weeks (b2) and 15 weeks (b3); nHACP group at 5 weeks (cl), at 10 weeks (c2) and at 15 weeks (c3); the fiber reinforced nHACP group at 5 weeks (dl), at 10 weeks (d2) and 15 weeks (d3), showing that only in the fiber reinforced group, could bone lacunas (red arrow area) and the bone cells (white arrow area) in the osseous callus be observed at 15 weeks after surgery, indicating that mature new bone has generated (Adapted with permission from Ref. . Copyright 2006 Elsevier Ltd)
Fig. 7.12 Histological appearances of the tissues of mouse subcutaneous dorsum where the material testing solutions were injected 2 weeks before. A lot of lymphocytes, mononuclear cells, and neutrophilic granulocytes could be observed in the poly (L-lacticacid) (PLA) and nanohydroxy- apatite/collagen (nHAC) groups, suggesting that serious foreign-body feedbacks have been caused by the two kinds of implants while the inflammatory cells was less than that in nanohydroxyapa- tite/collagen/PLA (nHACP) group, and no obvious foreign-body feedback happened in the fiber reinforced nHACP (nHACP/CF) group (Adapted with permission from Ref. . Copyright 2016 John Wiley & Sons)
bone implants represented that the collagen tissue formed between new bone and tricalcium phosphate particles. Therefore, with good biocompatibility and mechanical properties, carbon fiber reinforced tricalcium phosphate-polyvinyl alcohol filled polyester resin composites are capable of being used for space implantation of the large osteoperiosteal gap.
It have recognized that biomimetic nanofibrous scaffolds derived from natural biopolymers for bone tissue engineering applications require good mechanical and biological performances including biomineralization. Moreover, bone tissue engineering scaffolds are usually expected to be resorbable and to guide incoming progenitor and vascular cells until neotissue has formed . Zhou et al. successfully synthesized biodegradable rhBMP-2 loaded zein-based scaffolds with macroporous structures and silica (SBA-15) particles and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) fibers were incorporated into the scaffolds to produce an anti-infective composite scaffold suitable to osteogenic factor delivery in the functional repair of bone defects (Fig. 7.14) . By using the 3D micro-CT technique
Fig. 7.13 Roentgenographs of implanted bone after a different interval of periods: (a) 3rd day, 4th, 10th, 16th, 24th and 32nd weeks. (b) implanted bone. (c, d) Histopathology of tricalcium phosphate (purpfe)neocapillaiisation: showing asingle particle of and new bone formation around it (Adapted with permission from Ref. . Copyright 2007 Elsevier Ltd)
Fig. 7.14 SEM of
rhBMP-2 loaded silica/ hydroxypropyltrimethyl ammonium chloride chitosan (HACC)/zein composite scaffold (Adapted with permission from Ref. . Copyright 2014 Elsevier Ltd)
Fig. 7.15 (a) Wet, dry, and ash weight of ectopically formed bone induced by different composite caffolds after 4 weeks of implantation. (b) bone mineral density at 8 and 12 weeks. (c) Regenerated bone volume at both 8 and 12 weeks. (d) 3D Micro-CT reconstructed images of rabbit segmental radius at 4, 8, and 12 weeks with different implants: (a) pure zein-scaffold, (b) zein- hydroxypropyltrimethyl ammonium chloride chitosan (HACC), (c) zein-S20, and (d) zein-HACC- S20 scaffolds, indicating the zein-HACC-S20 scaffolds a significant improvementon the bone repair (Adapted with permission from Ref. . Copyright 2014 Elsevier Ltd)
and histological analysis, the zein-HACC-S20 (SBA-15, 20 wt%) scaffolds were found to significantly promote the bone repair in a rabbit model of critical size radial defects (Fig. 7.15), which demonstrated considerable promise for bone repair through tissue engineering.