Preparation of Nature Mimicked Composite Materials for Tissue Regeneration

Abstract

Skeletal defect is the second largest reason that leads to disability in worldwide. The bone is the most common transplanted tissue after blood. Bone regeneration strategy remains a significant challenge in the treatment. The filling of the lesion is an important factor to evaluate regenerative ability of implant along with its bioactivity. Most of regenerative commercial approaches are not very practical to use. Their application in trauma site caused varied degrees of damage to normal tissues. Pre-formed scaffolds cannot fill irregular defects as good as in situ forming implant. In-situ formed implants received great attention as they provided ease of use along with excellent healing capacity. A thermosensitive in situ forming hydrogels are synthesized by crosslinking of Chitosan (CS), Zein (ZN) and Hydroxyapatite (HA). CS, ZN and HA are biocompatible, economical, and biodegradable materials, used in range of biomedical applications. HA is gold standard biomineral used for excellent regeneration of bone tissue. ZN is a good source of amino acids that can help in regeneration of collagen matrix of hard and soft tissues. CS is the second most abundant natural biopolymer having excellent ability to from thermosensitive hydrogels. The major challenge of in situ forming implants is relatively low mechanical strength. To improve the mechanical strength and biological performance ZN is used. HA is synthesized via economical co-precipitation method. CS/HA/ZN thermosensitive hydrogel of different composition CS.HA.ZN (0), CS.HA.ZN (25), CS.HA.ZN (50) CS.HA.ZN (75) CS.HA.ZN (100) are prepared. Changing the pH of CS solution gradually acidic to neutral converted it to gel phase. Appropriate reagents that gradually change the pH are used. Here very less explored calcium glycerophosphate (Ca.GP) is used. Ca.GP help is contains Calcium and phosphate groups that are also component of hard tissue so presence of this also facilitate regeneration. Thermo sensitive in situ forming hydrogels are prepared by optimizing by changing the amount of neutralizing agent’s and Ca.GP and sodium bicarbonate (NaHCO3). CS, ZN and HA solution in the presence of neutralizing agents at 10 -12 °C existed in solutions form and constituted to hydrogels setting at 37 °C in 6-7 minutes. Gelation kinetics is monitored by test tube invert method. Prepared in situ hydrogels injectability, physical and biological tests are performed. Solutions of prepared hydrogel are conveniently injected drop wise from 18 Gauge needle that is frequently used for orthopedic and dental administrations. The addition of Zein improved the mechanical properties. The compressive strength of hydrogel CS.HA.ZN (0), CS.HA.ZN (25), CS.HA.ZN (50) CS.HA.ZN (75) and CS.HA.ZN (100) are 5.9, 8.47, 36.7, 20.2 and 52.2 MPa respectively. Swelling analysis of hydrogel showed good swelling property (50-55%) in PBS solution. Swelling is slightly increased with increasing the Zein content. Biodegradation analyses indicated that rapid degradation occur up to three days approximately 45% after that the gradual degradation rate is observed. Higher concentration of Zein decrease rate degradation. Prepared hydrogels are biocompatible, they showed good proliferation of NIH3T3 cell. Chance of infection is major reason of implant failure. In situ drug eluting grafts are best solution of this challenge. Drug carrying capacity of prepared hydrogels are monitored by using Metronidazole antibiotics. All samples showed 92-98% sustained drug delivery behavior. Prepared and drug loaded hydrogels showed effective antibacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus).