Authors in recent years, research is ongoing to determine the use of hydrogel as a scaffold for regenerative procedures. Various natural, synthetic and hybrid hydrogels are available but the use of natural agents deliver more promising results by exhibiting lower cytotoxic response . The ideal requirement for a scaffold to enhance the regeneration is to be biocompatible, having a neutral pH and should not induce inflammatory response . Furthermore, it should be able to promote bioactivity, namely cell adhesion, proliferation and migration . Although many studies have focused on the use of scaffold in tissue engineering, reports are scarce on the use of injectable carrageenan hydrogel scaffold infused with cissus quadrangularis in dentin-pulp complex regeneration. Hence, as a preliminary study, we focused on to assess the biocompatibility and antioxidant property of cissus quadrangularis incorporated carrageenan based hydrogel scaffold. A patent under the World intellectual property organization, with international publication no - WO 2008/081233 A2, with the inventor/applicant AVESTHA, GENGRAINE TECHNOLOGIES PVT LTD states that the percentage yield of whole plant extracts with active ingredients was the highest in water which was about 17.31%. The rest of the solvents used for extraction were hexane, 80% ethanol and acetone whose yields were much lesser than that of water. In the current study, this was the rationale of using aqueous extarct of Cissus quadrangularis for the preparation of hydrogel and further tests conducted .
In the present study the rationale for selecting the carrageenan hydrogel lies in the fact that it enhances favourable results in tissue regeneration process. This hydrogel has superior mechanical properties that depend on its molecular weight, source, concentration, type and degree of cross linkage . The molecular weight of the hydrogel has an influence on its degradation property. It has been found that as the molecular weight increases the degradation rate decreases . Studies showed that the degradation rate was higher when a lower weight scaffold such as chitosan-based hydrogels was used . Furthermore, the three dimensional structure of carrageenan has shown osteoblastic proliferation and adhesion . Combination of carrageenan hydrogels with different delivery system have shown successful outcome . The prerequisites for an injectable hydrogel include flowability under low pressure, rapid setting at the target site and preserving the appropriate integrity and strength . Due to its emulsifying and thixotropic property, it could be used as an injectable scaffold in enhancing the dentin-pulp regeneration .
In the present study, we have used a carrageenan based hydrogel incorporated with various concentrations of Cissus quadrangularis extracts which is a bioactive compound. To the best of our knowledge, the later has not been explored for its therapeutic potential in dentin-pulp regeneration; however, it potential for osteogenesis has been extensively studied. The osteogenesis potential of Cissus quadrangularis extract has been explored in various dental clinical situations such as periodontal bone regeneration , mandibular alveolar ridge distractions  and in maxillofacial  and mandibular fractures . In particular, the Cissus quadrangularis extracts have been shown to contain calcium, along with other compounds  and thus probably have shown to regulate osteoblastic activity  by enhancing osteoblastogenesis,  mineralization  and eventually induces bone formation for faster bone healing . Literature also showed that Cissus quadrangularis extract stimulated the mineralised nodules in dental pulp cells .
It's known that maxillofacial structures, including the bone and dentition, originate from the same embryologic origin from the neural crest and share similar compositions in terms of organic and inorganic components . Although, bone and teeth share a considerable amount of similarities, their developmental and regenerative properties differ due to their constructional proportions and their material and cellular compositions . The cells of both the tissues secrete an almost similar extracellular matrix, which is termed as predentin as osteoid respectively . The mineralisation of both dentin and bone extracellular matrix seemed to be initiated by a similar mechanism by the aid of matrix vesicles, later involving the secretion of families of specialised matrix proteins . There are various non-collagenous proteins such as bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) and osteopontin (OPN) that are not tooth-specific and found in both bone and dentin. They are expressed at varying levels and play a role in similar mineralisation mechanisms. As a result of their shared evolutionary history, bone and dentin have many characteristics in common. It's not surprising that the cells secrete matrices, osteoblasts and odontoblasts which are closely related cell types . Recent literature found the stage-specific and tissue expression of BSP , OPN  in reparative dentinogenesis. On the other hand, studies are showing the efficacy of Cissus quadrangularis extract on OPN,  bone morphogenetic protein (BMP)  and BSP  activation, which in turn gives us an idea on usage of this current material for dentin-pulp regeneration, as the material has immense potentiality for osteogenesis and mineralisation.
Another favourable property of Cissus quadrangularisis is its antibacterial property. In previous studies, in order to prevent the growth of residual endodontic bacteria, chitosan was used as a scaffold material . Cissus quadrangularis has demonstrated antibacterial efficacy against bacillus subtilis, pseudomonas aeruginosa, salmonella typhi, escherichia coli, proteus mirabilis, staphylococcus aureus and streptococcus pyogenes . Along with this it has been shown effective antibacterial effect against oral microorganisms such as S. mutans and L. Acidophilus .
In the present study, cissus quadrangularis and carrageenan hydrogels are combined to assess the antioxidant activity and biocompatibility. Till date there are no reports on the usage of combinations of these injectable hydrogels for assessing these properties in dentin-pulp regeneration.
For the assessment of the antioxidant activity, DPPH test was performed in the present study as it has been popularly used to test the antioxidant properties of the plant extracts. In the DPPH assay, the addition of the extract to a violet-colored DPPH solution reduces it to a yellow-colored product, diphenylpicryl hydrazine in a concentration-dependent manner. The convenience offered by the short duration of the assay, allow its wide applications to predict antioxidant activity . Dhanasekaran S et al. (2020) had evaluated the antioxidant property of cissus quadrangularis with different concentrations ranging from 25 to 400 µg/mL in ethanolic and methanolic extracts. In this in vitro model, the free radical scavenging activity was more in methanolic extract compared to ethanolic extract in a dose-dependent manner . In the present study, 10 and 20% w/v cissus quadrangularis showed a significant gradual increase in the antioxidant activity as compared to the control group, however a significant drop in the activity was observed with further increase in the concentration of the agent to 30% w/v. On SEM analysis it was observed that 10% and 20% w/v Cissus quadrangularis hydrogel showed an even distribution of the incorporated agent throughout the hydrogel but when the concentration is increased to 30% w/v, an uneven distribution with clumping of the Cissus quadrangularis particles was noted. This could be a probable reason for the reduced antioxidant activity seen with the increasing concentration.
Another crucial factor required in the micro-environment for successful tissue engineering is biocompatibility of the scaffold. To evaluate the same, carrageenan based hydrogel infused with Cissus quadrangularis was subjected to brine shrimp lethality assay. Current study showed a significant increase in the biocompatibility with the increasing concentration of the additive agent in the scaffold. Cissus quadrangularis extract, has been extensively explored for its antioxidant, anti-inflammatory and bone tissue regeneration in various in vitro studies (34). Previous reports have shown better cytocompatibility of Cissus quadrangularis when performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay for bone tissue engineering . In another study, least cytotoxicity of carrageenan hydrogels was seen when performed on L929 fibroblast cells . In the present study, Cissus quadrangularis and carrageenan hydrogels are combined to assess the antioxidant activity and biocompatibility. Till date there are no reports on the usage of combinations of these injectable hydrogels in dentin-pulp regeneration.
Limitations and Future directions
This is the preliminary in vitro study to assess the antioxidant and biocompatible property of carrageenan injectable hydrogel infused with Cissus quadrangularis for den-tin-pulp regeneration. The interaction between Cissus quadrangularis extract and carrageenan need to be studied further. Whether the sulphated groups and slightly acidic nature of the carrageenan inhibit the bio-active compounds from Cissus quadrangularis is unknown. Further studies need to be done using to stem cells from the apical papilla (SCAP) cell line to evaluate the biocompatibility of the hydrogel prepared. Studies need to be performed using organic and polar solvents to see if more active ingredients are released which can be used to synthesise a hydrogel with better antioxidant potential.