Comparative Early Local and Systemic Biochemical Host Response to Bovine Peritoneum-Derived Extracellular Matrix and Permacol in a Rat Abdominal Wall Repair Model

Abstract

Biological matrices are used in abdominal wall reconstruction, but their performance depends on the host response after implantation. We compared the early local and systemic biochemical responses to a newly developed bovine peritoneum-derived extracellular matrix (BPEM) and Permacol in a rat abdominal wall repair model. Sixty-four adult white outbred rats of both sexes were studied in a balanced 2 × 4 factorial design with independent terminal groups at postoperative days 5, 7, 14, and 21 (n = 8/material/time point). Peri-implant tissue and plasma from the same animal were analyzed for ASF, RNA, DNA, xanthine, H1, H2A-H3-H4, and H2B. Robust factorial models were used to assess the effects of material, time, and their interaction; exact Mann–Whitney tests with Holm correction identified time-specific between-material differences. Tissue showed significant time effects for all seven indicators and five material × time interactions, with the most pronounced between-material differences observed on day 5. Plasma showed several overall material effects but fewer time-specific differences. Significant post hoc differences were bidirectional rather than consistently higher with BPEM. These findings characterize the early local and systemic biochemical responses to BPEM and Permacol and extend the preclinical evaluation of the newly developed BPEM.

1. Introduction

Abdominal wall reconstruction is a major clinical application of implantable biomaterials, and implant selection can influence tissue repair, integration, and long-term function [1,2]. Biological matrices provide collagen-rich extracellular-matrix scaffolds that can support host tissue deposition and remodeling, but implantation also initiates inflammatory and foreign-body responses at the materialtissue interface [3,4,5,6]. In a nonhuman primate model of abdominal wall repair, Bolden et al. showed that local inflammatory markers, systemic immune measurements, and tissue-remodeling endpoints evolve over time and together provide a more complete description of the response to an acellular dermal matrix [7].

The magnitude and kinetics of the host response are influenced by matrix source and processing, including species, anatomical tissue origin, decellularization, collagen crosslinking, and sterilization [3,4,5,6,7]. Experimental abdominal-wall studies of dermal and porcine-derived matrices have further shown that tissue source and processing can influence inflammatory response, cellular infiltration, vascularization, remodeling, and mechanical behavior after implantation [8,9,10]. Recent experimental studies have also evaluated biological and implantable materials in regenerative and reconstructive settings, including their regenerative potential and biocompatibility [11,12]. This principle is also evident in a recent bioengineering study in which Pinni et al. evaluated an ovine extracellular-matrix-derived reinforced tissue matrix in a rat implant model and found changes in fibrosis- and inflammation-associated cellular markers despite no difference in total capsule thickness [13]. Together, these studies support evaluating biomaterials using an integrated pattern of host-response endpoints rather than a single isolated measurement.

Early biochemical changes may precede or accompany later structural remodeling and therefore provide complementary insight into host–biomaterial interactions. Measurements obtained directly from peri-implant tissue characterize the local microenvironment, whereas circulating measurements reflect the systemic component of the response. However, time-resolved comparisons of the same biochemical panel in peri-implant tissue and plasma after implantation of biological matrices remain limited. The biochemical panel was selected to characterize complementary processes associated with the early tissue response to implantation. ASF, together with RNA and DNA fractions, was used to characterize nucleic-acid precursor pools and nucleic-acid turnover; recent experimental work has likewise used ASF, extracellular nucleic acids, and histone proteins to characterize cellular and metabolic disturbance in rat plasma [14]. Histone-like fractions H1, H2A-H3-H4, and H2B were included to characterize chromatin-associated changes accompanying cellular and tissue injury [15,16], whereas xanthine was assessed as an indicator of purine catabolism and metabolic stress. Together, these parameters capture complementary biochemical aspects of the early tissue response to implantation.

BPEM is a decellularized bovine peritoneum-derived extracellular matrix that has previously been characterized in experimental studies at the structural, morphometric, biomechanical, and biological levels [17,18,19]. Permacol is an established cross-linked acellular porcine dermal collagen matrix with documented use in abdominal wall reconstruction and a substantial evidence base in the literature [8]. As a xenogeneic collagen-based biological matrix used for the same reconstructive indication, it was selected as a clinically and scientifically relevant comparator for BPEM. The objective of the present study was to extend the preclinical characterization of the newly developed BPEM by evaluating the temporal patterns of selected biochemical indicators in peri-implant tissue and plasma, using Permacol as an established biological comparator. We specifically evaluated whether the response depended on material, postoperative time, and their interaction, while treating each terminal time point as an independent group.

2. Materials and Methods

2.1. Biomaterials

The experimental implant was a bovine peritoneum-derived extracellular matrix (BPEM) obtained from bovine parietal peritoneum. Decellularized bovine peritoneum-derived matrix has previously been described and evaluated in experimental studies by Badyrov and colleagues [17,18]. In the present experiment, BPEM preparation included two sequential decellularization cycles in a solution containing 0.25% sodium dodecyl sulfate and 0.5% Triton X-100 for 3 h at room temperature with agitation at 200 rpm and intermediate washing steps. After decellularization, the samples were treated with RNase A (20 µg/mL) and DNase I (200 µg/mL) for 2 h at 37 °C and then washed again. Terminal radiation sterilization was performed at a dose of 16 kGy; this dose was selected because it preserved the morphological structure of collagen and elastin fibers. A comprehensive experimental characterization of BPEM was published previously [19]. Permacol (Covidien, Mansfield, MA, USA), a cross-linked acellular porcine dermal collagen matrix used for soft-tissue and abdominal wall reconstruction, served as the comparator.

2.2. Study Design

The study was designed to compare early biochemical responses to BPEM and Permacol at four independent terminal time points. Sixty-four rats were included in a balanced 2 × 4 design: two materials (BPEM and Permacol) and four postoperative time points (days 5, 7, 14, and 21), with eight animals in each material × time cell. Sex was not a prespecified stratification or allocation factor in the original experimental design. The distribution of male and female animals within each material × time-point group is provided in Supplementary Table S3. Because sex was not used as an allocation factor and each experimental cell included only eight animals, the study was not designed or adequately powered for sex-stratified analyses or for testing sex × material interactions. Each rat contributed peri-implant tissue and plasma at one predefined terminal time point only; the same animals were not followed longitudinally. Peri-implant tissue represented the local compartment, whereas plasma represented the corresponding systemic compartment. The same seven-indicator biochemical panel was measured in both compartments. Each animal was treated as the experimental unit (n = 1). The subgroup size of eight animals per material × time-point group was predefined within the original experimental program. A formal a priori power calculation was not performed because this was a pilot study and reliable prior data were unavailable to estimate the expected effect size and variability of the continuous biochemical outcomes. This approach is consistent with the methodological considerations described by Allgoewer and Mayer, who noted that formal sample-size estimation in pilot animal studies is often limited by insufficient prior information and that group sizes of five to six animals may be reasonable for continuous outcomes under common statistical assumptions [20]. No a priori criteria for animal or data exclusion were documented for this early-response dataset, and all 64 animals in the primary dataset were included in the analysis. Animals of comparable age and body weight were distributed equally among the experimental groups. Each animal received an individual identification number. Surgical and laboratory personnel were blinded to group identity and worked with coded data. No formal randomization was used. The experimental work was performed within the grant-funded research project of the Ministry of Education and Science of the Republic of Kazakhstan, “Development and Application of Xenoperitoneum Extracellular Matrix in the Surgical Treatment of Anterior Abdominal Wall Hernias,” state registration No. 0115RK00305. The rat abdominal wall defect model was selected because it permits standardized, controlled comparison of local material–tissue and systemic responses during preclinical evaluation of surgical biomaterials.

2.3. Animals and Animal Care

Adult white outbred laboratory rats (Rattus norvegicus) of both sexes were used. Each material × time-point group included four male and four female rats. Animals were 180 ± 15 days old and had a preoperative body weight of 187 ± 9 g. They were acclimatized for 14 days before the procedures and housed in the institutional vivarium at 18–22 °C under a 12 h light/12 h dark cycle, with a standard diet and free access to water. Invasive procedures were performed under general anesthesia and aseptic conditions. Routine postoperative analgesia was not administered because systemic analgesics can modulate immune, endocrine, and inflammatory responses and were therefore considered a potential pharmacological confounder for the biochemical and inflammatory endpoints evaluated in this study [21,22]. This approach formed part of the approved animal-use protocol and was specifically approved by the Bioethics Committee of Karaganda Medical University (Protocol No. 18, 16 May 2019). If predefined signs of postoperative pain or discomfort had occurred, additional analgesia with buprenorphine (0.05 mg/kg, subcutaneously, every 12 h for 48 h) would have been administered. Animals were monitored for general condition, activity, food and water intake, wound healing, postoperative complications, and signs of pain or distress. No animal required additional analgesia during the postoperative observation period. Humane endpoint assessment was based on these clinical indicators. Reporting was prepared with reference to the ARRIVE 2.0 guidelines [23].

2.4. Rat Abdominal Wall Repair Model

Surgery was performed under general anesthesia according to the standard operating procedures of the experimental program. General anesthesia was induced with diethyl ether by placing 3 mL of ether in a 3-L induction chamber for 5 min. To improve muscle relaxation and prolong anesthesia, xylazine hydrochloride (Romatar, 20 mg/mL) was administered intramuscularly according to the SOP. After hair removal, the ventral surface was treated with 2% chlorhexidine solution and 70% ethanol. An incision was made along the linea alba, and a 10 × 10 mm segment of the anterior abdominal wall was excised. The defect was repaired with BPEM or Permacol; the implant was fixed edge-to-edge to the margins of the anterior abdominal wall using interrupted or continuous over-and-over sutures with atraumatic suture material no larger than 4/0. After implant fixation, the wound surface was irrigated with sterile saline, hemostasis was checked, the wound was closed, and an aseptic dressing was applied.

2.5. Sample Collection

At each designated terminal time point, 3–5 mL of blood was collected by intracardiac puncture of the left ventricle under general anesthesia after thoracotomy. Blood was collected into EDTA-containing tubes and, within 30 min of collection, centrifuged for 15 min at 1000× g at 2–8 °C. The resulting plasma was separated and stored at −20 °C until biochemical analysis. A 10 × 10 mm sample of anterior abdominal wall tissue was excised immediately adjacent to the implant edge, rapidly frozen in liquid nitrogen, and mechanically pulverized in a mortar to a fine powdered consistency before subsequent biochemical analysis. For each biochemical analysis, 100 mg of pulverized peri-implant tissue was processed in 1.0 mL of extraction medium, corresponding to a fixed tissue-to-extractant ratio of 1:10 (w/v). The same tissue mass and extraction volume were used for all animals to ensure standardized sample preparation. Peri-implant tissue and plasma samples retained the same individual animal identifier, allowing paired interpretation of local and systemic responses within each terminal subgroup.

2.6. Biochemical Host-Response Assessments

Biochemical host-response assessments were performed in two biological compartments. Peri-implant tissue was used to assess the local response at the material–tissue interface, whereas plasma from the same animal was used to assess the systemic response. In both compartments, the panel included ASF, RNA, DNA, xanthine, and the histone-like protein fractions H1, H2A-H3-H4, and H2B. The nucleic-acid and histone-like fractions were operational measurements obtained by extraction and spectrophotometric fractionation. The panel was interpreted collectively as a biochemical profile of the early host response.

Sample comparability was ensured by the standardized tissue collection described in Section 2.5 and by the use of a fixed initial sample volume for plasma-based assays. Analytical quality control was ensured by applying the same sample-preparation and analytical procedures to all samples, including fixed reagent volumes, standardized incubation conditions, identical centrifugation parameters, and the same spectrophotometric measurement protocol.

2.6.1. Acid-Soluble Fraction, RNA, and DNA

The acid-soluble fraction (ASF), RNA, and DNA were determined by sequential acid fractionation according to L.I. Markusheva et al. [24]. For liquid samples, 1 mL of sample was mixed with 5 mL of 0.5 N perchloric acid and centrifuged at 5000 rpm for 15 min; the supernatant was used for ASF determination. The precipitate was then treated with 10 mL of 10% perchloric acid, incubated at 37 °C, and centrifuged again for 15 min at 5000 rpm; the resulting supernatant was used for RNA determination. The remaining precipitate was mixed with 3 mL of 10% perchloric acid, incubated for 7 min at 70 °C, and centrifuged under the same conditions; the supernatant was used for DNA determination. ASF and nucleic-acid hydrolysates were analyzed spectrophotometrically at 290 nm. The same sequential fractionation principle was applied to peri-implant tissue after the standardized tissue preparation described above. For peri-implant tissue, ASF, RNA, and DNA results are reported in arbitrary units per milliliter of standardized tissue extract (a.u./mL); plasma results are reported in a.u./mL.

2.6.2. Histone-like Protein Fractions

Histone-like protein fractions were determined by sequential extraction followed by spectrophotometry with biuret reagent at 330 nm [24]. For plasma, 2 mL of 5% perchloric acid was added to 1 mL of sample, the mixture was incubated for 1 h at 0–4 °C and centrifuged for 15 min at 1000 rpm; the supernatant was used to determine the H1 fraction. To isolate H2A-H3-H4, the remaining precipitate was mixed with 3 mL of an alcoholic solution, incubated for 18 h at 0–4 °C, and centrifuged again. The H2B fraction was obtained after adding 2 mL of 0.25 M hydrochloric acid to the remaining precipitate, incubating for 15 min at 0–4 °C, and centrifuging for 10 min. Peri-implant tissue was prepared as described above, after which the fractions were isolated according to the same sequential extraction principle. For peri-implant tissue, H1, H2A-H3-H4, and H2B results are reported in arbitrary units per milliliter of standardized tissue extract (a.u./mL); plasma results are reported in a.u./mL.

2.6.3. Xanthine

Xanthine was determined by direct UV spectrophotometry at a fixed wavelength of 276 nm according to the previously described method [25,26]. For plasma, 0.3 mL of sample was subjected to thermal coagulation in a boiling water bath for 5 min. After cooling, 3.0 mL of bidistilled water was added, followed by incubation for 30 min at 37 °C. The absorbance of the aqueous extract was measured at 276 nm against bidistilled water using a 10-mm optical-path cuvette. For peri-implant tissue, the aqueous extract of the standardized tissue preparation was analyzed using the same spectrophotometric principle. Bidistilled water served as the reference blank and corrected for background absorbance of the extraction medium and the instrumental baseline; however, this approach did not provide matrix-specific spectral correction or separation of co-absorbing compounds. Therefore, spectral interference from other endogenous UV-absorbing substances could not be completely excluded. No analyte-specific calibration curve or formal chromatographic validation against HPLC-UV or LC–MS/MS was performed. Accordingly, xanthine results were expressed in extinction units and interpreted as a method-dependent spectrophotometric index rather than as an analyte-specific absolute concentration.

2.7. Statistical Analysis

All analyses were based on individual-animal data, with each animal treated as the experimental unit. The study used a 2 × 4 factorial design with independent groups defined by material (BPEM vs. Permacol) and postoperative time (days 5, 7, 14, and 21). A separate sample of animals was analyzed at each terminal time point (n = 8 per material/time point); animals were euthanized at their designated time point for biological sample collection. Thus, postoperative time was treated as a between-subject factor rather than a repeated-measures factor, because the same animals were not examined sequentially across multiple time points. For each biochemical outcome, the main effects of material and postoperative time and the material × time interaction were evaluated using a full two-factor linear model with sum-to-zero contrasts. Sex was not included as a separate factor in the statistical analysis because it was not a prespecified stratification factor in the original experimental design. Type III robust Wald F tests were calculated with HC3 heteroscedasticity-consistent covariance estimates. Peri-implant tissue and plasma were analyzed separately. To identify time-specific between-material differences, post hoc BPEM-versus-Permacol comparisons were performed at each time point using exact two-sided Mann–Whitney U tests. For each biochemical outcome, the four time-specific comparisons were treated as one statistical family and adjusted using the Holm–Bonferroni procedure. The Holm–Bonferroni adjustment controlled multiplicity within each biochemical indicator across the four time-specific comparisons; no global multiplicity adjustment was applied across all seven biochemical indicators and both biological compartments. Therefore, the family-wise Type I error rate across the complete set of analyzed outcomes was not fully controlled. Rank–biserial correlation (r_rb) was reported as the pairwise effect-size measure. Because tissue and plasma were obtained from the same animal but represented biologically distinct compartments and were measured on different scales, their absolute values were not compared inferentially. Quantitative data are presented as median and interquartile range [Q1–Q3]. All tests were two-sided, and adjusted p < 0.05 was considered statistically significant. Statistical analyses were performed in Python 3.13.5 using SciPy 1.17.0 and statsmodels 0.14.6.

2.8. Use of Generative AI in Manuscript Preparation

During preparation of this manuscript, the authors used ChatGPT (5.6, San Francisco, CA, USA) for language editing and structural refinement. The authors reviewed and edited the output and take full responsibility for the content of the publication.

3. Results

3.1. Overall Pattern of the Early Biochemical Host Response

All 64 animals in the primary early-response dataset were included in the planned analysis, with n = 8 in each material × time cell. The factorial analysis showed that the early biochemical host response was influenced by both the implanted material and postoperative time, with the strongest time-dependent between-material differences observed in peri-implant tissue. In peri-implant tissue, postoperative time significantly affected all seven indicators, while material × time interactions were significant for ASF (p = 0.038), DNA (p < 0.001), H1 (p < 0.001), H2A-H3-H4 (p = 0.025), and H2B (p < 0.001). Significant overall material effects were detected for H2A-H3-H4 and H2B (both p < 0.001). In plasma, significant overall material effects were observed for RNA, DNA, xanthine, H1, H2A-H3-H4, and H2B, whereas significant material × time interactions were limited to H2A-H3-H4 and H2B. Complete factorial results are presented in Table 1.

Table 1. Robust factorial analysis of material, postoperative time, and material × time effects.

Compartment	Indicator	Material, p	Time, p	Material × Time, p
Tissue	ASF	0.769	0.007	0.038
Tissue	RNA	0.745	<0.001	0.487
Tissue	DNA	0.068	<0.001	<0.001
Tissue	xanthine	0.529	0.022	0.217
Tissue	H1	0.164	<0.001	<0.001
Tissue	H2A-H3-H4	<0.001	0.041	0.025
Tissue	H2B	<0.001	0.002	<0.001
Plasma	ASF	0.069	0.739	0.193
Plasma	RNA	0.030	0.501	0.254
Plasma	DNA	0.049	0.922	0.613
Plasma	xanthine	<0.001	<0.001	0.258
Plasma	H1	<0.001	0.073	0.319
Plasma	H2A-H3-H4	0.016	<0.001	<0.001
Plasma	H2B	0.046	0.185	0.007

Note: p values are from robust Wald F tests based on a full 2 × 4 linear model with HC3 heteroscedasticity-consistent covariance estimates. Degrees of freedom were 1 and 56 for the material effect and 3 and 56 for time and material × time effects. Significant p values (<0.05) are shown in bold.

3.2. Peri-Implant Tissue Response

The peri-implant tissue response showed the clearest differences between BPEM and Permacol. After Holm correction, eight tissue comparisons remained significant. Five of the seven indicators differed on day 5: ASF and H1 were higher with BPEM, whereas DNA, H2A-H3-H4, and H2B were lower. At later time points, the number of significant between-material differences decreased. H2A-H3-H4 remained lower with BPEM on days 7 and 21, and tissue xanthine was lower with BPEM on day 14. This pattern indicates that local biochemical differences were greatest during the earliest postoperative phase and became more limited thereafter.

3.3. Systemic Plasma Response

The systemic plasma response showed fewer time-specific between-material differences than the peri-implant tissue response. After Holm correction, three plasma comparisons remained significant. H2A-H3-H4 was lower with BPEM on day 5. On day 21, ASF was lower and H2B was higher with BPEM. The factorial model identified several overall material effects in plasma, whereas time-specific post hoc differences were less frequent than in peri-implant tissue.

3.4. Time-Dependent Material Differences Across Compartments

Taken together, the tissue and plasma analyses revealed compartment-specific host-response kinetics. Peri-implant tissue showed more pronounced time dependence and more material × time interactions, whereas plasma showed several overall material effects but fewer significant time-specific post hoc differences. The significant between-material differences were bidirectional rather than uniformly higher with BPEM. Thus, the response to BPEM did not show a persistent increase across the biochemical panel; instead, the magnitude and direction of the between-material differences varied with postoperative time and biological compartment. The comparative results are presented in Table 2.

Table 2. Time-specific BPEM-versus-Permacol comparisons remaining significant after Holm-Bonferroni correction.

Compartment	Day	Indicator	BPEM, Median [IQR]	Permacol, Median [IQR]	Holm-Adjusted p	r_rb
Tissue	5	ASF	0.438 [0.403–0.514]	0.286 [0.266–0.381]	0.028	0.781
Tissue	5	DNA	0.184 [0.170–0.214]	0.370 [0.321–0.374]	0.028	−0.781
Tissue	5	H1	1.558 [1.446–1.635]	1.091 [0.933–1.212]	<0.001	1.000
Tissue	5	H2A-H3-H4	0.804 [0.638–0.886]	1.598 [1.326–1.857]	0.004	−0.875
Tissue	5	H2B	0.937 [0.931–1.007]	1.980 [1.649–2.032]	<0.001	−1.000
Tissue	7	H2A-H3-H4	0.644 [0.596–0.754]	1.575 [1.293–1.691]	0.002	−0.938
Tissue	14	xanthine	0.337 [0.248–0.381]	0.470 [0.400–0.565]	0.019	−0.812
Tissue	21	H2A-H3-H4	1.091 [0.626–1.570]	1.771 [1.659–1.834]	0.003	−0.906
Plasma	5	H2A-H3-H4	0.199 [0.166–0.456]	1.418 [1.306–1.501]	0.001	−0.969
Plasma	21	ASF	0.207 [0.179–0.343]	0.449 [0.409–0.484]	0.012	−0.859
Plasma	21	H2B	1.549 [1.421–1.699]	0.863 [0.363–1.253]	0.028	0.781

Note: Holm-adjusted p values were calculated within each biochemical outcome across the four time-specific comparisons. Positive r_rb indicates higher ranks with BPEM; negative r_rb indicates lower ranks with BPEM. For peri-implant tissue, a.u./mL refers to arbitrary units per milliliter of standardized tissue extract; plasma values are reported in a.u./mL. Xanthine is reported in extinction units.

4. Discussion

This study characterized the early biochemical host response to a newly developed bovine peritoneum-derived matrix by directly comparing BPEM with Permacol. The main finding was that the biochemical response varied by both postoperative time and biological compartment: the most pronounced between-material differences occurred in peri-implant tissue during the earliest postoperative phase, whereas plasma differences were less frequent. Importantly, significant differences were bidirectional and did not show a persistent pattern of greater biochemical activation in the BPEM group. This overall response pattern provides a more informative basis for biomaterial evaluation than any single analyte considered in isolation.

The peri-implant compartment was the most sensitive to early between-material differences. All seven tissue indicators varied with postoperative time, and five showed significant material × time interactions. The clustering of five significant between-material differences on day 5, followed by only isolated differences on days 7, 14, and 21, suggests that material-associated differences were most pronounced during the earliest postoperative phase and became less distinct as postoperative adaptation progressed. This temporal pattern parallels the host-response framework used by Bolden et al., who interpreted local inflammatory and remodeling measurements according to their kinetics after implantation rather than as static endpoints [7].

The plasma findings complemented rather than reproduced the local tissue response. Several indicators showed overall material effects, but only three time-specific plasma comparisons remained significant after multiplicity correction. The divergence between local and circulating findings supports separate analysis of these compartments. In the Bolden study, local tissue cytokines and systemic implant-specific IgG also provided different but complementary information on the response to an acellular dermal matrix [7]. Our findings extend this local–systemic framework to an earlier postoperative interval by applying the same biochemical panel to tissue and plasma from each animal.

Among the individual markers, H2A-H3-H4 showed the most persistent material-associated differences, while H2B also showed significant material × time interactions in both compartments. These findings help characterize the biochemical pattern but should not be interpreted as evidence that a single histone-like fraction can serve as a standalone indicator of compatibility. These measurements were obtained using operational biochemical fractionation methods and are best interpreted together with ASF, nucleic-acid fractions, and xanthine as a composite profile of tissue turnover, cellular injury-related processes, and metabolic activity [14,15,16].

The broader biomaterials literature in bioengineering likewise supports an integrated interpretation of the host response. Pinni et al. reported that an ovine ECM-derived reinforced tissue matrix reduced macrophage and contractile-fibroblast markers without changing total capsule thickness, illustrating that favorable changes in biomaterial-associated response may occur even when a gross structural endpoint is unchanged [13]. In the present study, most time-specific BPEM-versus-Permacol comparisons were not significant after Holm correction, and the significant differences did not show a uniform direction. Overall, between-material differences varied by postoperative time and biological compartment and showed no consistent direction across the selected biochemical endpoints. These findings add a biochemical dimension to the existing structural, morphometric, biomechanical, and biological characterization of BPEM [17,18,19].

The strengths of the study include analysis of individual-animal data, a balanced 2 × 4 design with independent terminal cohorts, parallel assessment of the same biochemical panel in peri-implant tissue and plasma from the same animals, explicit modeling of material × time interactions, multiplicity-adjusted post hoc comparisons, and effect-size reporting. The main limitations are the operational rather than molecularly specific nature of the biochemical fractionation methods and the absence of a separate sham-operated defect-only group in the analyzed dataset. Direct UV spectrophotometry at a fixed wavelength does not provide chromatographic separation of individual compounds; therefore, spectral overlap from endogenous UV-absorbing substances and matrix effects cannot be completely excluded. Formal validation against HPLC-UV or LC–MS/MS was not part of the original analytical protocol. Xanthine measurements should therefore be interpreted within the limitations of the spectrophotometric method rather than as analyte-specific absolute concentrations. Although the four time-specific comparisons within each biochemical indicator were adjusted using the Holm–Bonferroni procedure, no global multiplicity correction was applied across all seven biochemical indicators and both biological compartments. Therefore, the possibility of false-positive findings across the overall set of analyses cannot be completely excluded, particularly for effects with p values close to the threshold for statistical significance. Such findings should be considered exploratory and require independent confirmation. Because sex was not a prespecified stratification factor and the study was not powered to assess sex-related effects, a potential contribution of sex-related biological variability to the observed biochemical differences cannot be completely excluded. As in other preclinical biomaterial studies, the present biochemical results should be integrated with histological, immunological, remodeling, and functional endpoints before drawing conclusions about overall biological compatibility [7,13]. Future studies should extend the observation period and combine biochemical kinetics with direct assessment of cellular infiltration, vascularization, matrix remodeling, and material performance. Because the study used a rat model, the biochemical response profile should not be directly extrapolated to human abdominal wall reconstruction. Future studies may also incorporate targeted functional immune-response assays [27].

5. Conclusions

In a rat abdominal wall repair model, the early biochemical responses to BPEM and Permacol varied according to postoperative time and biological compartment. The strongest between-material differences occurred in peri-implant tissue during the earliest postoperative phase, whereas fewer time-specific differences were detected in plasma. Significant differences were bidirectional and did not show a uniform pattern across the biochemical panel. These findings characterize the biochemical component of the early response to BPEM and Permacol and should be interpreted alongside histological, immunological, remodeling, and functional endpoints in further preclinical evaluation.