Macrophage-Marker Response and Exploratory Hepatorenal Safety of a Tricalcium Silicate–Biogenic Calcium Carbonate (Achatina fulica) Hybrid Pulp-Capping Material in a Mechanically Exposed Rat Molar Pulp Model: An In Vivo Study

Abstract

Objectives This study aimed to evaluate, in vivo, the macrophage marker response and exploratory hepatorenal safety of a complete candidate formulation of 75% tricalcium silicate (TCS) and 25% biogenic calcium carbonate (CaCO 3 ) from Achatina fulica shells, applied to mechanically exposed rat molar pulp, and compared with the manufacturer‐prepared commercial calcium silicate standard. Materials and Methods Forty‐eight male Wistar rats underwent mechanical pulp exposure of the maxillary first molars and were randomly allocated to three groups ( n = 16): negative control (saline), positive control (undiluted commercial calcium silicate cement, manufacturer‐hydrated, designated “100% TCS” for brevity), or the water‐activated hybrid (75% TCS + 25% A. fulica CaCO 3 ), with euthanasia on days 1, 3, 7, and 14 ( n = 4/subgroup). Characterization was performed using Fourier‐transform infrared spectroscopy (FTIR) and scanning electron microscopy with energy‐dispersive X‐ray spectroscopy (SEM‐EDX). The pulpal response was assessed by immunohistochemical cell counts for CD68 (pan‐macrophage), inducible nitric oxide synthase (iNOS) (M1‐associated), and CD163 (M2‐associated), and systemic safety by hepatorenal biochemistry (AST, ALT, BUN, creatinine) and blinded liver/kidney histopathology. The prespecified significance threshold was set at α = 0.05. Primary inference was drawn from between‐group comparisons pooled across the four time points ( n = 16 per group), with time‐point‐specific comparisons ( n = 4) reported descriptively. Results FTIR confirmed the presence of characteristic carbonate functional groups, and EDX indicated a calcium fraction of 23.7% on the analyzed surfaces, while chlorine decreased from 13.9% to 4.7% after hydration‐liquid substitution. Compared to the commercial standard, the hybrid was associated with lower pan‐macrophage infiltration (CD68) and M1‐associated marker (iNOS) counts at each timepoint and higher M2‐associated marker (CD163) counts, peaking at day 14 (all omnibus comparisons p < 0.001). Hepatorenal parameters and organ histopathology in the hybrid group were milder than those in the commercial standard group, although all four biochemical parameters remained above those of the operated saline control group, and hepatic portal inflammation was modestly higher in the hybrid group ( p = 0.009). Conclusions Compared with the commercial standard, the water‐activated 75:25 hybrid was associated with lower pro‐inflammatory (M1‐associated) and higher proresolving (M2‐associated) macrophage‐marker levels and with milder renal histopathology and minimal hepatic injury relative to the commercial standard. Because these associations reflect marker immunoreactivity in independent cross‐sectional samples rather than tracking individual cells, they support further preclinical evaluation of the hybrid for vital pulp therapy rather than proving efficacy. Because the formulations differed in more than one respect, the results reflect the net effect of the whole formulation rather than the isolated contribution of calcium carbonate.

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