Pre-mineralisation effect of nanobiocomposite bone scaffold towards bone marrow-derived stem cells growth and differentiation

Apatite layers formed by simulated body fluid (SBF) on the surface of calcium-based scaffolds have been proven to enhance the osteoblastic activity of pre-osteoblasts and osteogenic activity of bone marrow-derived stem cell (BM-SCs). Previously developed Alginate/Cockle shell powder nanobiocomposite bone scaffold (Alg/nCP) has been shown to possess excellent osteoconductive properties. The effect of pre-mineralization of the scaffold surface towards the growth and differentiation of BM-SCs’ were evaluated using microscopic and biochemical methods in scaffolds divided into SBF pre-treated and control groups at two time points. MTT proliferation assay showed statistically significant decrease in cell proliferation in SBF group for both culture periods. SEM observation revealed growth of BM-SCs and scaffold surface mineralisation and calcium deposition in both groups with higher intensity observable in the control group. Supporting biochemical studies showed a significant decrease in alkaline phosphatase (ALP) level indicating a lesser osteogenic differentiation in the SBF group as compared to control. Pre-mineralisation of scaffolds in SBF produced a contradicting result in which it did not provide a better environment for growth and proliferation of BM-SCs. However, the Alg/nCP scaffold did show potentials in supporting the osteogenic differentiation of the stem cells

Perbandingan antara perancah tulang nanobiokomposit alginat/ kulit kerang dan alginat/kalsium karbonat terhadap pertumbuhan osteoblas

Kalsium karbonat (CaCO3) memainkan peranan yang penting dalam merangsang pertumbuhan osteoblas. Kajian ini dijalankan untuk membandingkan prestasi perancah nanobiokomposit alginat/kulit kerang (nCP) yang mengandungi CaCO3 dari sumber semula jadi dan perancah alginat/kalsium karbonat (CC) yang mengandungi CaCO3 sintetik terhadap pertumbuhan osteoblas melalui kajian in vitro dan pemerhatian awal kebioserasian in vivo. Perancah tulang berbentuk tiga dimensi dibangunkan dengan menggunakan campuran 40% Alginat dan 60% serbuk kulit kerang bersaiz nano (perancah nCP) atau serbuk CaCO3 sintetik (perancah CC). Kajian in vitro terhadap pembebasan kalsium dan aktiviti enzim alkalin fosfatase (ALP) pada kedua-dua perancah yang telah dibenihkan dengan osteoblas ditentukan pada hari ketiga, kelima dan ketujuh pengkulturan. Kajian in vivo dijalankan dengan implantasi subkutan perancah yang telah dibenihkan dengan osteoblas pada bahagian dorsum lapan ekor mencit selama 21 hari. Setelah 21 hari, perancah dikeluarkan dari mencit untuk pemerhatian histologi menggunakan pewarnaan H&E and von Kossa. Hasil kajian in vitro menunjukkan peningkatan secara signifikan (p < 0.05) perembesan kalsium dan aktiviti enzim ALP pada perancah nCP pada hari ketujuh berbanding perancah CC pada hari ketiga dan kelima. Pemerhatian histologi terhadap kedua-dua perancah menunjukkan infiltrasi dan proliferasi osteoblas serta pembentukan tisu tulang peringkat awal. Pembentukan saluran darah juga dapat dikenal pasti pada perancah nCP. Kedua-dua perancah menunjukkan potensi untuk menyokong dan membantu pertumbuhan osteoblas namun perancah nCP didapati menunjukkan potensi yang lebih baik secara keseluruhan. Kesimpulannya, CaCO3 dari sumber semula jadi iaitu kulit kerang dan bersaiz nano berpotensi untuk dijadikan sebagai biobahan di dalam aplikasi kejuruteraan tisu tulang

Fabrication of ciprofloxacin loaded alginate/cockle shell powder nanobiocomposite bone scaffold

Orthopedic implant infection is one of the most challenging issues in bone tissue engineering industry. Hence, local delivery of antibiotics incorporated into a fabricated bone scaffold possibly provides a more rapid bacteria inhibitory effect. In this study, pure ciprofloxacin loaded alginate/cockle shell powder nanobiocomposite bone scaffolds are fabricated with 5 wt% and 10 wt% ciprofloxacin respectively and tested for drug encapsulation, drug release and antibacterial properties towards common implant infecting bacterial strains (Staphylococcus aureus and Pseudomonas aeruginosa). Results from the studies showed a low drug encapsulation and drug release regardless of the concentration of drugs loaded with no significant differences noted (p<0.05). However, bacterial inhibition studies through direct contact and using eluted samples from drug release studies showed some inhibitory effects towards the growth of both bacterial strains tested. These findings were further justified with microscopy observations on biofilm and bacterial colony formation. Mineralization studies conducted additionally indicated that the scaffolds characteristics was not compromised due to drug loading. Although pure ciprofloxacin may not be the most suitable antibiotic to be incorporated into the nanobiocomposite bone scaffold, the study did provide some insight to the possible use of the scaffold for future drug delivery applications

Nano-cockle shell powder and alginate as novel injectable bone filler: A preliminary formulation and characterization study

Cockle shells of the Anadara granosa species consist of 95-99% aragonite form of calcium carbonate (CaCO3) crystals. Aragonites are known to be denser which allows it to be incorporated, resolved and replaced by bone tissues over time when used in bone tissue grafting. An injectable biomaterial-based bone filler with appropriate injectability and biological properties was formulated using powdered cockle shells in nanoscale (nCSP) and sodium alginate (Alg). Initial attempts in formulating the bone filler using alginate and citric acid as a setting agent in the composition of nCSP: Alg of 60: 40, 70: 30 and 80: 20 wt.% produced variable results in the bone filler characterization. Characterization study of nCSP-Alg bone filler with 70:30 wt.% compositions showed excellent injectability (p<0.05), viscosity (p<0.05) and anti-washout ability that was further evaluated through physicochemical analysis, morphology and biocompatibility studies. Scanning electron micrographs revealed plate-like nanocrystal deposits with micropores ranging between 1.5 – 7.4 μm. XRD and FTIR evaluation indicated the presences of peaks associated with aragonite form of CaCO3. Biocompatibility studies with MG63 osteoblast showed osteoconductivity of the bone fillers with excellent cell adherence, growth and subsequent mineralization of the matrices. In conclusion, nCSP-Alg biomaterial-based bone filler with 70:30 wt.% compositions shows promising use as a cost effective bone grafting material for clinical application in the field of bone tissue engineering

Cardamonin attenuates hyperalgesia and allodynia in a mouse model of chronic constriction injury-induced neuropathic pain: possible involvement of the opioid system

Neuropathic pain arises from the injury of nervous system. The condition is extremely difficult to be treated due to the ineffectiveness and presence of various adverse effects of the currently available drugs. In the present study, we investigated the antiallodynic and antihyperlagesic properties of cardamonin, a naturally occurring chalcone in chronic constriction injury (CCI)-induced neuropathic pain mice model. Our findings showed that single and repeated dose of intra-peritoneal administration of cardamonin (3, 10, 30 mg/kg) significantly inhibited (P<0.001) the chronic constriction injury-induced neuropathic pain using the Hargreaves plantar test, Randall-Selitto analgesiometer test, dynamic plantar anesthesiometer test and the cold plate test in comparison with the positive control drug used (amitriptyline hydrochloride, 20 mg/kg, i.p.). Pre-treatment with naloxone hydrochloride (1 mg/kg, i.p.) and naloxone methiodide (1 mg/kg, s.c) significantly reversed the antiallodynic and antihyperalgesic effects of cardamonin in dynamic plantar anesthesiometer test and Hargreaves plantar test, respectively. In conclusion, the current findings demonstrated novel antiallodynic and antihyperalgesic effects of cardamonin through the activation of the opioidergic system both peripherally and centrally and may prove to be a potent lead compound for the development of neuropathic pain drugs in the future

Development and evaluation of novel alginate/cockle shell powder nano-biocomposite porous 3D scaffold for bone repair

Shells are comparable to bones of vertebrates due to the similarities in mechanical properties and strength. The cockle shell material may act as an anolog of calcium carbonate in an in vivo condition that makes it a potential bone grafting material. The present study involves the development and evaluation of a novel three-dimensional alginate/nano cockle shell powder biocomposite bone scaffold prepared through lyophilization and divalent cation cross-linking methods. Element analysis revealed that the shell material consisted of 96% of calcium carbonate with no traces of toxic elements while physiochemical analysis revealed a predominantly aragonite form of calcium carbonate polymorph. The cockle shell powder was converted to nano particles using a biomineralization catalyst through a simple chemical method and was used as a representative of the inorganic phase while sodium alginic acid (alginate) was used as the organic phase in the development of the nano-biocomposite scaffold. The scaffold mixture was prepared in varying composition ratios, characterized and evaluated through various characterization studies. Scanning electron microscopy (SEM) analysis revealed the micro architecture of the scaffolds with pore size ranging from 10 – 336 μm diameters. The porosity of the scaffolds was found to be above 60%. Mechanical properties of the tested scaffolds showed the composition ratio of 40% alginate and 60% nano cockle powder (Alg:nCP=40:60) possesed favorable mechanical properties ranging between the spongy bone structures compressive strength. Swelling ratio of the scaffolds showed an average of 30% medium uptake ability with 20 – 30% changes in diameter. Enzymatic degradation test revealed an increase in structural stability proportional to the amount of nano cockle shell powder within the composition while pH changes observed during degradation studies revealed a neutralizing effect of nano cockle shell powder towards the potential acidification of the solution during alginate degradation. The physiochemical properties of the materials and the subsequent chemical interactions evaluated revealed the phase purity of the materials as well as the scaffolds ionic interaction characteristics contributing to an increase in thermal stability. In-vitro studies conducted on MG63 human osteoblast-like cells revealed good biocompatibility and absences of cytotoxic effect of the scaffolds with higher cell viability noted in scaffolds of 40:60 ratio and was used for further in-vitro evaluation. Cell growth and adherence towards the scaffold materials were evaluated for a period of 48 hours, 7 and 14 days using SEM, Element Detection System (EDS) and histological evaluation. The results showed good attachment and spreading properties of the cells within 48 hours and were found to have grown into large cell clusters by Day 7 with distinctive presence of calcium nodules that was verified using EDS analysis on the nano-biocomposite scaffolds. At Day 14, a completely mineralized scaffold structure was observed in the nano-biocomposite scaffolds supported by findings from EDS analysis that showed the presence of phosphate and calcium as well as histological observations showing presence of osteoid like tissues.In-vivo analysis of the scaffold implanted in a 5 mm osseous defect at the proximal part of the left tibia bone of New Zealand White rabbits revealed evidence of better healing quality of the nano-biocomposite scaffolds compared to control scaffolds as well as empty unfilled defects that were created simultaneously on the right proximal tibia bone of the animals. The quality of healing assessed after seven weeks post implantation through histomorphometric evaluations at three different depths of the defects revealed a significantly better healing in the nano-biocomposite defect site at all three sections compared to the empty defect site as well as with the lower section of the control scaffold defect site. Comparatively, the regeneration of bone tissues were found to occur in a systematic coordinated way with larger areas of matured bone tissues observed in the presences of the nano-biocomposite scaffolds. The remaining void spaces within the defect sites with implants were found to be significantly lesser compared to those of the empty defects while the amount of remaining nano-bicomposite scaffold material was found to be significantly higher compared to the control scaffolds at all three regions evaluated. Statistical analysis for all data’s were done using One-way Analysis of Variance (ANOVA) followed by the post-hoc Tukey’s test, unless otherwise stated, where p<0.05 was accepted as significant. As a conclusion, the developed nano-biocomposite scaffold using alginate and nano cockle shell powder was found to show promising results to be used in the field of bone tissue engineering. The scaffolds showed good porous architectures that enhance its osteoconductive properties by facilitating better and faster bone regeneration in addition to being completely biocompatible as well as a cost effective alternative for bone grafting in the near future

Cytotoxicity and oxidative stress evaluation of Alginate/Cockle Shell powder nanobiocomposite bone scaffold on osteoblast

Biocompatibility and growth of osteoblast on bone scaffolds play an important role towards their therapeutic application. The presence of oxidative stress generated by bone scaffolds highly influences osteoblast growth and its functional performance. In this study in-vitro interaction of developed Alginate/Cockle Shell powder nanobiocomposite bone scaffold on osteoblast with regards to cytotoxicity and oxidative stress are evaluated. Cytotoxicity studies using MTT assays revealed a significant increase in viability of cultured osteoblast in the presences of the scaffold extracts. The growth of osteoblast on the scaffold were not deterred with the presence of any major oxidative stress factors as determined through oxidative stress profile studies using SOD, GSH and ROS assays. The nanobiocomposite scaffold evaluated in this study shows promising use in regards to facilitating osteoblast proliferation, growth and viability

Vancomycin loaded alginate/cockle shell powder nanobiocomposite bone scaffold for antibacterial and drug release evaluation

Bacterial infection and biofilm formation is a major concern in orthopaedic implants and bone reconstructive surgery complications that may be addressed with localized drug delivery system. The potential use of a fabricated nanobiocomposite bone scaffold using alginate and nano cockle shell powder for drug release and antibacterial properties was investigated. Vancomycin loaded bone scaffolds were fabricated with 3 and 5 wt% vancomycin, respectively, while a non-drug loaded scaffold was used as controls. The mineralization of the scaffolds using simulated body fluid (SBF) as well as biofilm formation were evaluated using microscopic observations. Drug release study and antimicrobial activity of the eluent from each sampling period was tested for growth inhibition of Staphylococcus aureus and Staphylococcus epidermidis for a period of 21 days. Significant difference of cumulative amount of vancomycin eluted from scaffolds loaded with 5 wt% vancomycin compared to 3 wt% (p<0.05) were noted. Eluent from both groups showed inhibitory effect against bacterial strain tested for 21 days. The findings are further supported with histological observations of reduced biofilm formation by Staphylococcus epidermidis on surface of 5 wt% vancomycin loaded scaffolds compared to control scaffolds. Basic mineralization studies conducted showed no alteration in drug loaded scaffolds characteristics compared to control scaffolds. Findings from this study indicates antibacterial properties can be conferred to the fabricated bone scaffold with successful incorporation of vancomycin with potentials to be used for local drug delivery application

Development and Characterization of Novel Porous 3D Alginate-Cockle Shell Powder Nanobiocomposite Bone Scaffold

A novel porous three-dimensional bone scaffold was developed using a natural polymer (alginate/Alg) in combination with a naturally obtained biomineral (nano cockle shell powder/nCP) through lyophilization techniques. The scaffold was developed in varying composition mixture of Alg-nCP and characterized using various evaluation techniques as well as preliminary in vitro studies on MG63 human osteoblast cells. Morphological observations using SEM revealed variations in structures with the use of different Alg-nCP composition ratios. All the developed scaffolds showed a porous structure with pore sizes ideal for facilitating new bone growth; however, not all combination mixtures showed subsequent favorable characteristics to be used for biological applications. Scaffolds produced using the combination mixture of 40% Alg and 60% nCP produced significantly promising results in terms of mechanical strength, degradation rate, and increased cell proliferation rates making it potentially the optimum composition mixture of Alg-nCP with future application prospects

Zerumbone alleviates neuropathic pain through the involvement of L-arginine-nitric oxide-cGMP-K+ ATP channel pathways in chronic constriction injury in mice model

The present study investigates the involvement of the l-arginine-Nitric Oxide-cGMP-K⁺ ATP pathways responsible for the action of anti-allodynic and antihyperalgesic activities of zerumbone in chronic constriction injury (CCI) induced neuropathic pain in mice. The role of l-arginine-NO-cGMP-K⁺ was assessed by the von Frey and the Randall-Selitto tests. Both allodynia and hyperalgesia assessments were carried out on the 14th day post CCI, 30 min after treatments were given for each respective pathway. Anti-allodynic and antihyperalgesic effects of zerumbone (10 mg/kg, i.p) were significantly reversed by the pre-treatment of l-arginine (10 mg/kg), 1H [1,2,4]Oxadiazole[4,3a]quinoxalin-1-one (ODQ), a soluble guanosyl cyclase blocker (2 mg/kg i.p.) and glibenclamide (ATP-sensitive potassium channel blocker) (10 mg/kg i.p.) (p < 0.05). Taken together, these results indicate that systemic administration of zerumbone produces significant anti-allodynic and antihyperalgesic activities in neuropathic pain in mice possibly due to involvement of the l-arginine-NO-cGMP-PKG-K⁺ ATP channel pathways in CCI model