The focus on adoption of biomimetic mineralization in fabricating bone tissue scaffolds has surged owing to the numerous benefits it holds. Mimicking the natural mechanisms of the bone promotes better osteo-integration subsequently aiding cell adhesion, proliferation and enhanced regeneration of the bone tissue. Along these lines, a wide selection of natural polymers, organic/non-organic hybrids and their composites have been critically explored. Among every such combination, inorganic hydroxyapatite (HA) stands afront due to its significant role in biological systems. The natural bone tissue principally is composed of collagen fiber mineralized with nano-HA crystals.
Biomineralization using microorganisms is advantageous for the production of nanoparticles of HA as it presents the unique ability to control the structure, orientation and surface morphology while generating nano HA crystals of uniform shape and size, essential parameters such as wettability, biocompatibility, non-toxicity and good osteo-conductivity. More importantly, these nano-sized HA contributes to higher bioactivity and aid osteoblasts in synthesis of alkaline phosphatase. Alkaline phosphatase is the key enzyme involved in the bacterial synthesis of hydroxyapatite production. However, certain properties such as weak tensile strength, brittleness and limited biochemical properties require more research for improved development of nanocomposites of HA.
The present study aims to explore sustainable biomaterials for HA synthesis via a biomimicry route. Gram-negative bacterium Serratia marcescens NCIM5246 (SHA) and Gram-positive bacterium Bacillus tequilensis MCC4228 (BHA) were utilized for deposition of HA using defined biomineralization media. Characteristic HA groups, low crystallinity, phase purity, B-type carbonation, uniform agglomeration of needle-like HA nanocrystals with 20-50 nm and 30-60 nm length and breadth of 5-10 nm and 3-10 nm for SHA and BHA, respectively was recorded. Calcination carried out at higher temperatures for SHA and BHA depicted resolved peaks and increased crystallinities. Furthermore, greater than 80% cell viability was witnessed along with deposition of essential trace ions such as Zn, Sr, Mg, Na and K by bacteria.
To effectively participate towards environmental sustainability is now a priority for every industry alike. Biomedicine accommodates the criteria on the upstream as well as downstream sectors via incorporation of biowastes to produce precursors as well as in preparation of scaffolds. In this vein, the novelty of our study lies in utilization of biowastes namely egg shells (ESHA) and SCOBY pellicle (SNHA), from the food and beverage industry for bacterial synthesis of HA. Physicochemical, structural and biological properties were thoroughly determined using various techniques wherein egg shell derived CaCl2 was deployed as precursor in biomineralization media for HA synthesis. Results indicated high similarity with natural bone parameters in terms of high carbonate content, low crystallinity, preferential c-axis growth producing needle-like morphologies of 20-60 nm length and 3-10 nm breadth, trace ion distribution and cell viability of greater than 90%. Distinctly, Si was deposited in ESHA rendering beneficial traits.
Further, SNHA scaffolds were prepared using two novel routes including agar plate (SNHA-A) and liquid broth method (SNHA-B) of HA deposition by Serratia marcescens NCIM5246. Results clearly depicted the deposition of HA on both the surface as well as infiltration into nanocellulosic 3D fibrous network of SN. The overlapping of characteristic cellulose and HA functional groups also confirmed HA deposition with B-type carbonation, poor crystallinity with densely agglomerated rod-shaped HA nanocrystals of high thermal stability. Critically, SNHA-B outweighed SNHA-A in terms of higher HA deposition, thermal stability with mineral phase greater than 50% and relatively higher resolved peaks. It is also well known that despite nanocellulosic HA nanocomposites possessing advantageous traits; there are not absorbed by the human body due to lack of cellulase enzymes. In order to enhance in vivo bioabsorbability, oxidation was performed using sodium periodate to produce OSN, OSNHA-A and OSNHA-B membranes. Characterization data illustrated that oxidation treatment did not significantly influence inherent 3D nanofibrous structure of SN, however reflected reduced crystallinity from 90% in SN to 84% in OSN. Typical HA nanocrystals were depicted similar to physiological bone processes particularly penetrating into the OSN structure intercalated with cellulose nanofibers. Moreover, thermal stability was found to decrease when compared to SNHA-A and SNHA-B with 14-15% mineral phase constitution of OSNHA-A and OSNHA-B indicating influence of oxidation also validated by presence of hemiacetal bonds. In vitro cytocompatibility assays and osteogenic activity tests showed that all SN and OSN scaffolds are non-toxic with greater than 97% cell viability and promote osteointegration. In particular, OSNHA-B reflected the highest degradation rate of 62.5% with increased ALP activity in comparison with SN, OSN and respective A and B methods of HA deposited membranes.
Finally, in order to holistically mimic the natural bone structure an attempt was made to synthesize ion-substituted and co-substituted HA nanoparticles. When bio-mineralization process was done in presence of ions such as Sr, Zn and Mg there was restriction of crystal size and also formation of complex 3D architecture. When varying concentration of Sr, Zn and Mg ions (Sr-SHA, Zn-SHA and Mg-SHA) were substituted characteristic HA peaks were seen,with B-type carbonation, large agglomerated nanocrystals of high phase purity and poor crystallinity similar to SHA. The increase in Sr, Zn and Mg % on SHA also confirmed effective substitution of Ca2+ by the selected ions. Whereas, in the case of co-substituted HA (1% Sr Mg Zn SHA) significant alterations or peak shifting was not witnessed owing to lower concentrations investigated. Yet, crystallinity presented changes with respect to lattice parameters and cell volume. Strontium displayed highest cell volume of to 531.07 A3 greater than SHA of 526.4 A3. In general, decreased a and increased c-axis could enhance solubility and biofunctionality. Bacterial deposition of other cations and anions such as Na+, K+ and Cl− ions was found to be intact similar to SHA. The dimensions of length 40-70 nm and breadth 4-10 nm and ion content were found to be comparable to natural bone. Results presented successful substitutions of 1% Sr Mg Zn SHA in Ca2+ sites with greater than 90% cell viability that eventually renders greater osteoconductive properties with high biocompatibility for bone tissue engineering. Till date, no study has highlighted this combination of ions (Sr, Zn and Mg) individually or co-substituted in bacterial synthesized HA.
Ultimately, analytical results validate the potential of researched membranes and materials for bacterial HA deposition. Bacterial synthesis occurs at ambient temperatures, is eco-friendly in nature, simplicity and uniform distribution of HA nanocrystals. It is thereby evident that from a sustainable and competent view, bacterial synthesized HA nanocomposites are a clear considerable choice and successful biomimicry was achieved with bone-like characteristics thus reflecting its effectiveness for bone defect repair.
Publications:

PATENT:
1. Patent granted on 01/08/2022 “Microbially synthesized Nanocomposite”. (Patent number- 402779)

II. REFEREED JOURNAL PUBLICATIONS
1. Paramasivan, M., Kumar, T. S. S., & Chandra, T. S. (2022). Microbial Synthesis of Hydroxyapatite-Nanocellulose Nanocomposites from Symbiotic Culture of Bacteria and Yeast Pellicle of Fermented Kombucha Tea. Sustainability, 14(13), 8144. https://doi.org/10.3390/su14138144
2. Paramasivan, M., Kumar, T. S., Kanniyappan, H., Muthuvijayan, V., & Chandra, T. S. (2023). Microbial biomineralization of hydroxyapatite nanocrystals using Bacillus tequilensis. Ceramics International, 49(4), 5621-5629. https://doi.org/10.1016/j.ceramint.2022.10.138
3. Paramasivan, M., Kumar, T. S., Kanniyappan, H., Muthuvijayan, V., & Chandra, T. S. (2023). Biomimetic Ion Substituted and Co-Substituted Hydroxyapatite Nanoparticle Synthesis Using Serratia Marcescens. Scientific Reports, 13, 4513 https://doi.org/10.1038/s41598-023-30996-z