Introduction:Osteoarthritis is one of the most degenerative diseases that affect the hyaline cartilage of the knee. The most affected individuals are the old people who are over 60, and athletes may encounter such injury leading to osteoarthritis if not treated properly. Moreover, many endeavors have been established to treat defected cartilage.
However, some of them prove its ability to heal the cartilage, but other procedures have failed.Tissue engineering has opened a new hope. The aim of tissue engineering is to replace or facilitate the growth of injured tissue, by coming material scaffold, cells and growth factor. Scaffold offers a 3D structure environment for the cells to attach, grow and differentiate. Scaffold improvement to achieve optimal tissue regeneration environment is being searched. One of the most challenges is designing a scaffold with control release system without affecting the physical, chemical and mechanical properties of the scaffold.Literature review: A degenerative joint disease which known as osteoarthritis is characterized by cartilage degeneration and the overgrowth of osseous tissues. Osteoarthritis prevalence increases with age, and it is the most common disease affected older people 1.
Joint pain, swelling, tenderness, and stiffness are the common symptoms that affect osteoarthritis patients and disturb their lives 2. OA is among the most challenging joint diseases, due to the lack of self-healing of articular cartilage 3. Bones that make up the joints in the body, their ends covered with articular cartilage which is typically hyaline cartilage. Hyaline cartilage confers the joints its smooth and frictionless movement. Furthermore, it scatters the stress on joints that induced by body weight.
Anatomically, articular cartilage is avascular tissue and lacking neural cells too. This type of tissue is primarily composed of chondrocyte and extracellular matrix of collagen type 2 and proteoglycans 4. Osteoarthritis pathogenesis characterized by losing of collagen and proteoglycan, which hens, disrupt the extracellular structure and impair biomechanical properties of cartilage tissue 5, 6. Chondrocyte on another hand, encounters apoptotic death. It has been noticed that the proliferation of chondrocyte is somewhat activated, but the process is unachievable 7, due to the production of matrix-degrading enzymes such as, matrix metalloproteinase 13 (MMP13) that degrade collagen, and metalloproteinase with thrombospondin motifs-5 (Adamts-5) which target aggrecan during osteoarthritis pathogenesis 8, 9.Repairing cartilage damage resulting from osteoarthritis or trauma still a medical issue. Many methods of treatments have been introduced, encompassing advantage and disadvantage, but reaching a complete repair procedure is being an unmet goal. Tissue engineering provides a new choice for cartilage repair.
This method incorporates cells, biomaterial scaffold and growth factor 10.Scaffolds for tissue engineering are being designed and serve as microenvironment 3D matrix. In cartilage tissue engineering, scaffolds can serve cartilage’s cells the adhesion, proliferation, and differentiation 11. Moreover, they mediate cell to cell signaling and interaction. There is a growing interest and desire to mimic the complexity of natural tissue, for that, the physical and biochemical property of used scaffold in cartilage regeneration is crucial 12.
Leakage of cells, poor cell survival, poor cell differentiation, inadequate integration into the host tissue, incorrect distribution of cells, and dedifferentiation of the normal cartilage are the common problems in tissue engineering 13, 14.Cell survival and differentiation can be enhanced by improving the physical, mechanical and biochemical properties of the scaffold. For that, the ideal scaffold should be biodegradable, give way to newly formed tissue and should not hamper the growth of newly formed cartilage or induce any inflammatory response. Furthermore, several important aspects must be taken into consideration, including porosity, elasticity, surface energy, chemical functionality, responsiveness to pH or temperature, micro- and Nano-topography of surface and ability to be metabolized in the body when biodegraded 15.
In addition, the scaffold should also favor cell migration and should support the biomechanical environment of native cartilage in-vivo 16.Stimulate the growth of new hyaline cartilage tissue and repair injury can be acquired by incorporating a tunable drug delivery mechanism into scaffolds. Moreover, cells recruitment, adhesion, proliferation, and differentiation in a scaffold can be facilitated by Addition of drugs that have an influence on cellular function and tissue regeneration 17.
There is a growing desire to delivering multiple drugs simultaneously, sequentially, or in multiphasic patterns. Researchers hypothesized that the speed, quantity, and quality of tissue regeneration, will be enhanced over time by control release of drugs. Controlled drug delivery can be accomplished by physically or chemically adsorbing the drug onto scaffold’s surface, encapsulating the drug directly within the scaffold, or by incorporating Micro/Nanoparticles drug delivery systems on the scaffold. Drug release can occur by diffusion or the degradation of a scaffold or the delivery system 18.The quantity and duration of drug released from scaffold can be controlled by altering the composition of the material, delivery system, or the methods of drug integration. In addition, kinetics and duration of drug release can also be tuned by changing the drug dose or by coating the material with a substrate (e.g. heparin) that specifically or nonspecifically binds cells or drugs 19.
The challenges in the field of controlled release lie in the ability to finely control the release of the drug without affecting the mechanical or structural properties of the scaffold and without damaging or quickly eluting the drug itself 18.Methodology:Synthesis of succinyl -ChitosanChitosan will be synthesized according to an already reported procedure slightly modification 20. 0.5g of chitosan highly viscous, medium molecular weight and low molecular weight will be dissolved in 5% (v/v) lactic acid solution and then methanol will be added to dilute the solution as (1:40 v/v). 0.75 (w/v) of succinic anhydride will be added to the mixture and will be stirred at room temperature for 24h. The mixture will be precipitated by adjusting the solution pH to 6~7.
The precipitate will be re-dissolved in H2O and dialyzed for 3 days. The purified product will be freeze-dried and stored at 4°C.Synthesis of aldehyde- HAHA will be synthesized according to an already published procedure with slight modification 21.0.01 % (w/v) HA at different molecular weight 200, 500 and 1000 KD will be dissolved in 100mL d.d H2O. 0.05% v/v aqueous solution of 0.
5M of sodium periodate will be added dropwise, and the reaction will be stirred for 2h at room temperature in the dark. 0.01% (v/v) of Ethylene glycol will be added and will be stirred for 1h. The solution will be dialyzed against H2O for 3 days, and freeze-dried.Preparation of hydrogels compositeS-CS and A-HA will be dissolved in H2O separately at different concentration. The crosslinked hydrogels will be formed by mixing CS and HA solutions at room temperature. Various volume ratios between CS and HA will be carried on to achieve better composite ratio. The gelation time and porosity of composite hydrogels will be monitored 22.
Preparation of Microsphere encapsulated of IGF-1/TGF?3A total of 250 mg of PLGA will be dissolved into 1 ml of di-chloromethane. Insulin-like growth factor-1(IGF-1) or transforming growth factor beta TGF?3 will be diluted and added to the PLGA solution, forming a mixture (primary emulsion) that will be emulsified for 1 min (w/o). The primary emulsion will be then added to 2 ml of 1% poly-vinyl alcohol (PVA; molecular weight, 30,000 to 70,000), followed by 1-min mixing (w/o/w).100 ml of PVA will be added and stirred for 1 min at room temperature. A total of 100 ml of 2% isopropanol will be added to the final emulsion and continuously stirred for 2 hours to remove the solvent. Empty microspheres will be used as Control.
To Incorporation of microsphere into the hydrogel, Microspheres encapsulating IGF-1 or TGF?3 will mix as (1:1 v/v) and mix directly with hydrogels at different volume ratio and will be stirred at room temperature to achieve gelation 23.Release kineticRelease kinetics of IGF-1 and TGF?3 from PLGA microsphere and hydrogel incorporated microspheres will be measured by incubating microspheres encapsulating IGF-1 or TGF?3 or hydrogels separately in PBS, bovine serum albumin or media at 37°C with mild agitation for interval times. At the end of incubation times, Microspheres or hydrogels will be centrifuged at 2500 rpm for 5 min, supernatant will be collected. Released IGF-1 and TGF?3 will be measured by using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s protocols.Characterization of hydrogels composite: MorphologiesSurface and porosity of hydrogels will be characterized by utilizing scanning electron microscopy (SEM) after gelation. The hydrogels will be freeze-dried and then gold-coated.Infrared (IR) spectroscopic measurementFTIR spectra of chitosan, HA and hydrogel composites will be measured to confirm the expected functional groups after polymer modification and hydrogel formation. Equilibrium swellingThe known weights of freeze-dried hydrogels will be immersed in DMEM with10%FBS and PBS, respectively, and incubated at 37°C for 2h until equilibrium of swelling will be reached.
The hydrogels will be removed and immediately weighed with a microbalance after excess media is removed by filter paper. The equilibrium swelling ratio (ESR) will be calculated by using the following equation: ESR= (Ws – Wd)/Wd Ws is weights of the hydrogels at the equilibrium swelling state Wd is weights of the hydrogels at dry stateCompressive ModulusHydrogel solution will be injected into a 12-well culture plate for 15 min to obtain columned hydrogels (22mm diameter, 6mm height).Compressive modulus of elasticity will be measured by using a dynamic mechanical analyzer in unconfined compression at room temperature.Degradation in vitroDegradation of hydrogels will be examined with respect to weight loss. Initially weighed hydrogels will be incubated in PBS or media at 37°C, and monitored. At specified interval times, hydrogels will be removed from the PBS or media and weighed. The weight loss ratio will be defined as 100%×(W0?Wt )/W0. The weight remaining ratio will be defined as1-100%×(W0?Wt )/W0.
(W0) The initial weight (Wt) The weight after incubationStatistical analysis Data from all the experiments will be analyzed by using Analysis of Variance (ANOVA). Statistical significance will be set to p-value ? 0.05. Results will be presented as the mean ± standard deviation.