Yonggang Yan

Synthesis and characterization of nano-HA/PA66 composites

Based on the bioactivity and biocompatibility of hydroxyapatite (HA) and the excellent mechanical performance of polyamide 66 (PA66), a composite of nanograde HA with PA66 was designed and fabricated to mimic the structure of biological bone which exhibits a composite of nanograde apatite crystals and natural polymer. The HA/PA66 composite combines the bioactivity of HA and the mechanical property of PA66. This study focused on the preparation method of HA/PA66 composite and the influence of HA crystals on the characterization of the composite. HA slurry was used directly to prepare HA/PA66 composite by a solution method, in which HA is able to form hydrogen bond, i.e. chemical bonding with PA66. The nano-HA needle-like crystals treated by hydrothermal method are better in the particle size distribution and the particle dispersion. The morphology, crystal structure and crystallinity as well as crystal size of these needle-like crystals are similar to bone apatite. The nano-HA needle-like crystals dispersed uniformly in PA66 matrix with reinforcement effect and can prevent the micro-crackle spreading into cleft and fracture during the deformation process. The mechanical testing shows that the nano-HA/PA66 composite has a good mechanical property, and may be a promising bone replacement material.

Analyzing the behavior of a porous nano-hydroxyapatite/polyamide 66 (n-HA/PA66) composite for healing of bone defects.

The aim of this study was to analyze the behavior of the porous nano-hydroxyapatite/polyamide 66 (n-HA/PA66) composite grafted for bone defect repair through a series of biological safety experiments, animal experiments, and a more than 5-year long clinical follow-up. The biological safety experiments, carried out in accordance with the Chinese Guo Biao and Tolerancing (GB/T)16886 and GB/T16175, revealed that porous n-HA/PA66 composite had no cytotoxicity, no sensitization effect, no pyrogenic reaction, and that its hemolysis rate was 0.59% (less than 5%). Rabbit models of tibia defects with grafted porous n-HA/PA66 composite were established. After 2 weeks, the experiment showed that osteogenesis was detected in the porous n-HA/PA66 composite; the density of new bone formation was similar to the surrounding host bone at 12 weeks. After 26 weeks, the artificial bone rebuilt to lamellar bone completely. In the clinical study, a retrospective review was carried out for 21 patients who underwent serial radiographic assessment after treatment with porous n-HA/PA66 composite grafts following bone tumor resection. All wounds healed to grade A. No postoperative infections, delayed deep infection, nonspecific inflammation, rejection, or fractures were encountered. At a mean follow-up of 5.3 years, the mean Musculoskeletal Tumor Society’s (MSTS) 93 score was 29.3 points (range: 28–30 points) and mean radiopaque density ratio was 0.77±0.10. The radiologic analysis showed that porous n-HA/PA66 composite had been completely incorporated with the host bone about 1.5 years later. In conclusion, this study indicated that the porous n-HA/PA66 composite had biological safety, and good biocompatibility, osteoinduction, and osseointegration. Thus, the porous n-HA/PA66 composite is an ideal artificial bone substitute and worthy of promotion in the field.

In vitro degradability, bioactivity and primary cell responses to bone cements containing mesoporous magnesium–calcium silicate and calcium sulfate for bone regeneration

Mesoporous calcium sulfate-based bone cements (m-CSBC) were prepared by introducing mesoporous magnesium–calcium silicate (m-MCS) with specific surface area (410.9 m² g−1) and pore volume (0.8 cm³ g−1) into calcium sulfate hemihydrate (CSH). The setting time of the m-CSBC was longer with the increase of m-MCS content while compressive strength decreased. The degradation ratio of m-CSBC increased from 48.6 w% to 63.5 w% with an increase of m-MCS content after soaking in Tris–HCl solution for 84 days. Moreover, the m-CSBC containing m-MCS showed the ability to neutralize the acidic degradation products of calcium sulfate and prevent the pH from dropping. The apatite could be induced on m-CSBC surfaces after soaking in SBF for 7 days, indicating good bioactivity. The effects of the m-CSBC on vitamin D3 sustained release behaviours were investigated. It was found that the cumulative release ratio of vitamin D3 from the m-CSBC significantly increased with the increase of m-MCS content after soaking in PBS (pH = 7.4) for 25 days. The m-CSBC markedly improved the cell-positive responses, including the attachment, proliferation and differentiation of MC3T3-E1 cells, suggesting good cytocompatibility. Briefly, m-CSBC with good bioactivity, degradability and cytocompatibility might be an excellent biocement for bone regeneration.

Degradable biocomposite of nano calcium-deficient hydroxyapatite-multi(amino acid) copolymer.

Background and methods A nano calcium-deficient hydroxyapatite (n-CDHA)-multi(amino acid) copolymer (MAC) composite bone substitute biomaterial was prepared using an in situ polymerization method. The composition, structure, and compressive strength of the composite was characterized, and the in vitro degradability in phosphate-buffered solution and preliminary cell responses to the composite were investigated. Results The composite comprised n-CDHA and an amide linkage copolymer. The compressive strength of the composite was in the range of 88–129 MPa, varying with the amount of n-CDHA in the MAC (ranging from 10 wt% to 50 wt%). Weight loss from the composite increased (from 32.2 wt% to 44.3 wt%) with increasing n-CDHA content (from 10 wt% to 40 wt%) in the MAC after the composite was soaked in phosphate-buffered solution for 12 weeks. The pH of the soaking medium varied from 6.9 to 7.5. MG-63 cells with an osteogenic phenotype were well adhered and spread on the composite surface. Viability and differentiation increased with time, indicating that the composite had no negative effects on MG-63 cells. Conclusion The n-CDHA-MAC composite had good cytocompatibility and has potential to be used as a bone substitute.

Preparation and properties of BSA-loaded microspheres based on multi-(amino acid) copolymer for protein delivery.

A multi-(amino acid) copolymer (MAC) based on ω-aminocaproic acid, γ-aminobutyric acid, L-alanine, L-lysine, L-glutamate, and hydroxyproline was synthetized, and MAC microspheres encapsulating bovine serum albumin (BSA) were prepared by a double-emulsion solvent extraction method. The experimental results show that various preparation parameters including surfactant ratio of Tween 80 to Span 80, surfactant concentration, benzyl alcohol in the external water phase, and polymer concentration had obvious effects on the particle size, morphology, and encapsulation efficiency of the BSA-loaded microspheres. The sizes of BSA-loaded microspheres ranged from 60.2 μm to 79.7 μm, showing different degrees of porous structure. The encapsulation efficiency of BSA-loaded microspheres also ranged from 38.8% to 50.8%. BSA release from microspheres showed the classic biphasic profile, which was governed by diffusion and polymer erosion. The initial burst release of BSA from microspheres at the first week followed by constant slow release for the next 7 weeks were observed. BSA-loaded microspheres could degrade gradually in phosphate buffered saline buffer with pH value maintained at around 7.1 during 8 weeks incubation, suggesting that microsphere degradation did not cause a dramatic pH drop in phosphate buffered saline buffer because no acidic degradation products were released from the microspheres. Therefore, the MAC microspheres might have great potential as carriers for protein delivery.

Degradability and cytocompatibility of tricalcium phosphate/poly(amino acid) composite as bone tissue implants in orthopaedic surgery

In this study, a tricalcium phosphate (TCP) and poly (amino acid) copolymer (PAA) biocomposite were fabricated for bone repair and characterized. The results show that the compressive strength of the TCP/PAA composites increased with an increase in the TCP content at TCP contents less than 40 w%. The weight loss of the composite after soaking in phosphate buffered saline for 12 weeks significantly increased with an increase in the TCP content, revealing its good degradability. In addition, the composite maintained adequate mechanical strength during the degradation period because it underwent a surface erosion process. In vitro MG63 cell culture experiments showed that the composite is non-cytotoxic and thus allows cells to adhere, proliferate and differentiate. Osteoid formation was evidenced on the composite surfaces 12 weeks after its implantation into the femoral bone of dogs. Furthermore, the composite combined directly with the host bone tissue without fibrous capsule tissue, and no inflammatory responses were found, showing the good biocompatibility of the composite. It is expected that the composite may be used for the development of bone implants for orthopaedic surgery.

Wollastonite nanofiber-doped self-setting calcium phosphate bioactive cement for bone tissue regeneration.

The purpose of this study was to synthesize a self-setting bioactive cement by incorporation of wollastonite nanofibers (WNFs) into calcium phosphate cement (CPC). The composition, morphology, setting time, compressive strength, hydrophilicity, and degradation of WNF-doped CPC (wnf-CPC) were investigated. Scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and inductively coupled plasma atomic emission spectroscopy were utilized. Additionally, methyl-thiazolyl-tetrazolium bromide assay, scanning electron microscopy, inductively coupled plasma atomic emission spectroscopy, and histological evaluation were used to study the cell and tissue responses to wnf-CPC, both in vitro and in vivo. The results confirmed that the addition of WNFs into CPC had no obvious effect on the setting time or the compressive strength of wnf-CPC, provided the WNF amount was not more than 10 wt%. However, the hydrophilicity and degradability of wnf-CPC were significantly improved by the addition of WNFs – this was because of the change of microstructure caused by the WNFs. The preferred dissolution of WNFs caused the formation of microporosity in wnf-CPC when soaked in tris hydrochloride solution. The microporosity enlarged the surface area of the wnf-CPC and so promoted degradation of the wnf-CPC when in contact with liquid. In addition, MG-63 cell attachment and proliferation on the wnf-CPC were superior to that on the CPC, indicating that incorporation of WNFs into CPC improved the biological properties for wnf-CPC. Following the implantation of wnf-CPC into bone defects of rabbits, histological evaluation showed that wnf-CPC enhanced the efficiency of new bone formation in comparison with CPC, indicating excellent biocompatibility and osteogenesis of wnf-CPC. In conclusion, wnf-CPC exhibited promising prospects in bone regeneration.

Mechanics, degradability, bioactivity, in vitro, and in vivo biocompatibility evaluation of poly(amino acid)/hydroxyapatite/calcium sulfate composite for potential load-bearing bone repair.

A ternary composite of poly(amino acid), hydroxyapatite, and calcium sulfate (PAA/HA/CS) was prepared using in situ melting polycondensation method and evaluated in terms of mechanical strengths, in vitro degradability, bioactivity, as well as in vitro and in vivo biocompatibility. The results showed that the ternary composite exhibited a compressive strength of 147 MPa, a bending strength of 121 MPa, a tensile strength of 122 MPa, and a tensile modulus of 4.6 GPa. After immersion in simulated body fluid, the compressive strength of the composite decreased from 147 to 98 MPa for six weeks and the bending strength decreased from 121 to 75 MPa for eight weeks, and both of them kept stable in the following soaking period. The composite could be slowly degraded with 7.27 wt% loss of initial weight after soaking in phosphate buffered solution for three weeks when started to keep stable weight in the following days. The composite was soaked in simulated body fluid solution and the hydroxyapatite layer, as flower-like granules, formed on the surface of the composite samples, showing good bioactivity. Moreover, it was found that the composite could promote proliferation of MG-63 cells, and the cells with normal phenotype extended and spread well on the composite surface. The implantation of the composite into the ulna of sheep confirmed that the composite was biocompatible and osteoconductive in vivo, and offered the PAA/HA/CS composite promising material for load-bearing bone substitutes for clinical application.

Effects of mechanical loading on the degradability and mechanical properties of the nanocalcium-deficient hydroxyapatite–multi(amino acid) copolymer composite membrane tube for guided bone regeneration

Background and methods Guided bone regeneration (GBR) is a new treatment for bone defects, and the property of membrane is critical to the success of GBR. This study focuses on a novel membrane tube for GBR, which was prepared by a nanocalcium-deficient hydroxyapatite–multi(amino acid) copolymer (n-CDHA-MAC) composite. The biomechanical strength and degradability of this membrane tube under mechanical loading after immersion in phosphate-buffered solution were investigated to evaluate the effects of mechanical loading on the membrane tube. The membrane-tube group with no mechanical loading and femora bone were used as controls. Results The compressive strength and bending strength of n-CDHA-MAC membrane tubes were 66.4 ± 10.2 MPa and 840.7 ± 12.1 MPa, which were lower than those of the goats’ femoral bones (69.0 ± 5.5 MPa and 900.2 ± 17.3 MPa), but there were no significant (P > 0.05) differences. In the in vitro degradability experiment, all membrane tubes were degradable and showed a surface-erosion degradation model. The PH of solution fluctuated from 7.2 to 7.5. The weight and mechanical strength of loaded tubes decreased more quickly than nonloaded ones, with significant differences (P < 0.05). However, the strength of the loaded group after degradation achieved 20.4 ± 1.2 MPa, which was greater than the maximum mechanical strength of 4.338 MPa based on goat femoral middle stationary state by three-dimensional finite-element analysis. Conclusions n-CDHA-MAC membrane tubes have good biomechanical strength during degradation under mechanical loading. Therefore, this membrane tube is an ideal GBR membrane for critical size defects of long bones in goats for animal experiments.

In vivo biocompatibility of new nano-calcium-deficient hydroxyapatite/poly-amino acid complex biomaterials.

Objective To evaluate the compatibility of novel nano-calcium-deficient hydroxyapatite/poly-amino acid (n-CDHA/PAA) complex biomaterials with muscle and bone tissue in an in vivo model. Methods Thirty-two New Zealand white rabbits were used in this study. Biomaterials were surgically implanted into each rabbit in the back erector spinae and in tibia with induced defect. Polyethylene was implanted into rabbits in the control group and n-CDHA/PAA into those of the experimental group. Animals were examined at four different points in time: 2 weeks, 4 weeks, 12 weeks, and 24 weeks after surgery. They were euthanized after embolization. Back erector spinae muscles with the surgical implants were examined after hematoxylin and eosin (HE) staining at these points in time. Tibia bones with the surgical implants were examined by X-ray and scanning electron microscopy (SEM) at these points in time to evaluate the interface of the bone with the implanted biomaterials. Bone tissues were sectioned and subjected to HE, Masson, and toluidine blue staining. Results HE staining of back erector spinae muscles at 4 weeks, 12 weeks, and 24 weeks after implantation of either n-CDHA/PAA or polyethylene showed disappearance of inflammation and normal arrangement in the peripheral tissue of implant biomaterials; no abnormal staining was observed. At 2 weeks after implantation, X-ray imaging of bone tissue samples in both experimental and control groups showed that the peripheral tissues of the implanted biomaterials were continuous and lacked bone osteolysis, absorption, necrosis, or osteomyelitis. The connection between implanted biomaterials and bone tissue was tight. The results of HE, Masson, toluidine blue staining and SEM confirmed that the implanted biomaterials were closely connected to the bone defect and that no rejection had taken place. The n-CDHA/PAA biomaterials induced differentiation of a large number of chondrocytes. New bone trabecula began to form at 4 weeks after implanting n-CDHA/PAA biomaterials, and lamellar bone gradually formed at 12 weeks and 24 weeks after implantation. Routine blood and kidney function tests showed no significant changes at 2 weeks and 24 weeks after implantation of both biomaterials. Conclusion n-CDHA/PAA composites showed good compatibility in in vivo model. In this study, n-CDHA/PAA were found to be safe, nontoxic, and biologically active in bone repair.