Characterization of host response, resorption, and strength properties, and performance in the presence of bacteria for fully absorbable biomaterials for soft tissue repair

A non-contaminated hernia defect model with subcutaneous mesh implantation was approved by the Institutional Animal Care and Use Committee at CBSET, Inc. (Lexington, MA). All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals. The study consisted of 60 (n = 60) male Sprague–Dawley rats (332.7–483.6 g, ~8 weeks old), divided into 2 groups, including fully resorbable synthetic [Gore^® Bio-A^® Tissue Reinforcement (Bio-A^®; n = 30)] and fully resorbable biologically derived [Phasix™ Mesh (Phasix™; n = 30)]. The structural differences between these biomaterials are depicted in scanning electron micrographs in Fig. 1. Phasix™ is comprised of a monofilament, macroporous design of poly-4-hydroxybutyrate derived from genetically modified K12 E. coli bacteria, whereas Bio-A^®is comprised of a multifilament, microporous, sheet-like design of poly(glycolide:trimethylene carbonate).

Meloxicam (1 mg/kg, PO or SQ) was administered on the morning of implantation, and isoflurane gas inhalant anesthesia was delivered at 0.25–5% in oxygen, to effect. Buprenorphine (0.02 mg/kg, SQ) was also administered at the time of anesthetic induction. The animal was then placed in dorsal recumbency, and the surgical site was shaved, prepared, and draped aseptically. Using sterile technique, a 3–5-cm midline incision was made through the skin of the ventral abdomen (Fig. 2a). The skin on the right side of the abdomen was bluntly dissected to reveal the subcutaneous plane. A 0.5-cm defect was created in the muscle layer using a surgical scalpel without penetrating the peritoneal cavity (Fig. 2b, c). A 2 × 2 cm square piece of mesh was soaked/rinsed according to the instructions for use (as appropriate) and then implanted into the subcutaneous plane and fixed over the surgical defect using four interrupted 5-0 Prolene™ sutures, one at each corner (Fig. 2d). The skin was closed with continuous 4-0 Vicryl™ sutures in a subcuticular fashion, followed by skin staples or wound clips. Animals were monitored during recovery until upright and ambulatory. Meloxicam (1 mg/kg, PO) was administered every 24 h for the first three postoperative days. Co-Flex^® or ELASTIKON^® wrapping material was used to prevent disruption of the surgical incision site. Animals were euthanized at 2, 4, 8, 12, 16, or 24 weeks (n = 5 animals of each mesh type at each time point) via carbon dioxide asphyxiation in accordance with the American Veterinary Medical Association (AVMA) guidelines.

Following euthanasia, the skin over the implant site was reflected, and the implant site was photographed, excised, and immersion-fixed in 10% neutral, buffered formalin. Specimens were then processed, embedded in paraffin, sectioned at 5 µm, and stained with Masson’s trichrome and hematoxylin and eosin. Inflammatory response was scored by a blinded, board-certified veterinary pathologist as described previously [7, 8]: 0 = no response, 1 = minimal/barely detectable, 2 = mild/slightly detectable, 3 = moderate/easily detectable, and 4 = marked/very evident.

Morphometric analysis was also conducted on the Masson’s Trichrome stained slides for one representative section per implant. At low magnification, a 6-mm segment of the implant was selected and evaluated for repair thickness, which was defined as the sum of the mesh thickness and the surrounding collagenous material, measured in millimeters (mm). Repair thickness measurements were taken at approximately 0.5 mm increments along the length of the cross section, resulting in a total of 13 (n = 13) measurements per section. The contribution of the thickness of the mesh itself to these measurements was also assessed on three (n = 3) measurements per section. Repair thickness and mesh thickness measurements are reported below as mean ± standard deviation.

An established rabbit bacterial inoculation model was utilized for the current study [7‐9]. Approval was obtained from the Institutional Animal Care and Use Committee at WuXiAppTec, Inc. (St. Paul, MN). All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals. The study consisted of 20 male New Zealand White Rabbits (2.9–3.7 kg), divided into 2 groups, including fully resorbable synthetic [Bio-A^® Tissue Reinforcement (Bio-A^®; n = 10)] and fully resorbable biologically derived [Phasix™ Mesh (Phasix™; n = 10)].

As described previously [7, 8], animals were initially anesthetized with 2.5–5% inhalational isoflurane (to effect), and maintained at 0.5–5% (to effect) throughout the procedure. The dorsal area was prepared for aseptic survival surgery by shaving the entire midline region of the back, cleaning the operative area with three alternating scrubs of povidone-iodine/70% isopropyl alcohol solutions, and then sterile surgical drapes were placed over the entire field. Following preparation of the dorsal surface for aseptic surgery, a 2.0–2.5-cm lateral incision along the dorsal surface of the thoracic spine at the level of the scapulae was created through the dermal layer. A single incision was made parallel to the midline of the back, cutting through the fascia and exposing the paravertebral muscle. The fascial membrane was incised with a scalpel and enlarged using blunt dissection to create a pocket for implanted mesh. The implant pockets were created bilaterally along the lateral wall toward the lateral aspect of the scapula of each rabbit to accommodate insertion and placement of one test device (3.8-cm-diameter circle) per side into the subcutaneous space. One previously die-cut and soaked/rinsed (as appropriate according to the instructions for use for each device) implant was gently introduced into each of the subcutaneous pockets (Fig. 3a). Each animal received the same device in each of the bilateral pockets. The distal needle of a Vacutainer^® blood collection set was then removed to create a bacterial injection catheter. The 1.5-mm-diameter tubing was placed into each pocket between the device and the infraspinous fossa of the scapula. Through blunt dissection, a hemostatic clamp was used to create a tunnel from each implant pocket towards the caudal aspect, parallel to the midline, for approximately 7–10 cm. The injection catheter tubing was retracted through this tunnel so that one end was in the implant pocket, and the other end was pulled through an incision made at the other end of the tunnel. This permitted remote inoculation of each pocket once the implantation procedure was complete. The tubing was temporarily secured with a purse–string suture. The pocket surrounding each device was sutured closed using a continuous pattern, reconnecting the subcutaneous tissues around the device. The cutaneous tissues were closed with a two-layer closure technique using absorbable sutures. Staples were also used to close the incision sites. Each device/isolated pocket was then inoculated with clinically isolated MRSA (1.0 ml of 1 × 10⁸ CFU/ml). Prepared syringes were used to deliver the determined dose into each pocket via the indwelling inoculation catheters, each followed by a 1.0 ml flush of sterile saline from separate syringes (Fig. 3b). The catheters were subsequently removed and previously placed bilateral purse–string sutures were closed to seal each pocket. Animals were recovered from anesthesia and allowed free access to food and water. At 7 days post-implantation, euthanasia was achieved by intravenously injecting rabbits with 150 mg/kg of sodium pentobarbital, in accordance with the American Veterinary Medical Association (AVMA) Panel of Euthanasia. Following euthanasia, the overlying skin and adipose tissue were removed from the dorsal aspect of each animal aseptically, and implant locations were dissected to expose each device.

Upon explantation, each device and device location was inspected for evidence of abscess formation. As described in previous studies [7, 8], abscess was independently scored by a trained investigator at the study site as: none (0), mild (1), moderate (2), or marked (3). The device from the left side of the animal was further analyzed for MRSA colony-forming units (CFU) within or adherent to the device, and the device from the right side of the animal was analyzed for mechanical (ball burst) strength.

Photographs were taken of each implant site at each time point during necropsy, and representative images are shown in Fig. 4. The rapid resorption of Bio-A^® is clearly evident at the later time points, particularly at 16 and 24 weeks, compared to the relatively unchanged appearance of the Phasix™ implants throughout the duration of the study.

As shown in Fig. 5, Phasix™ implant sites exhibited a median inflammation score of 2.0 at alltime points evaluated, representing a mild inflammatory response that remained consistent over time. Macrophages (mild) and lymphocytes (minimal to negligible) were consistent at all time points. Neutrophils and granulomas were minimal at 2 weeks and progressively decreased to negligible or absent at all subsequent time points. Eosinophils were minimal up to 8 weeks, progressively decreased thereafter, and were absent at 24 weeks. Giant cells decreased from mild at 2 weeks to minimal-mild at all subsequent time points.

Bio-A^® sites exhibited a median inflammation score of 4.0 at 2 and 4 weeks, indicating a marked inflammatory response. However, between 8 and 24 weeks, the median score for Bio-A^® sites ranged from 2.0–3.0, indicating a transition to a mild/moderate inflammatory response over time, upon bulk material resorption. Macrophages and eosinophils decreased progressively over time, with macrophages transitioning from moderate-marked to minimal-mild and eosinophils transitioning from mild-moderate to absent. Neutrophils decreased from mild at 2 weeks to negligible or absent at all subsequent time points. Giant cells progressively decreased from moderate-marked at 2 weeks to minimal at 24 weeks. Lymphocytes (minimal-mild) remained consistent over time.

No significant differences in inflammatory response were observed between materials except at 4 weeks when Bio-A^® (median 4.0, interquartile range 3.5–4.0) demonstrated significantly greater inflammation compared to Phasix™ (median 2.0, interquartile range 1.5–2.5, p < 0.01).

Mesh thickness (mm) and repair thickness (mesh thickness + surrounding fibrotic response, mm) for Phasix™ and Bio-A^® sites are depicted in Figs. 6 and 7 (mean ± standard deviation). The pre-implantation thickness (T ₀) of Phasix™ has been previously reported as 0.51 mm (0.02 in) and is depicted as a dashed line in Fig. 7a [10]. As shown in the white bars of Fig. 7a, the thickness of Phasix™ mesh remained similar to its pre-implantation value and was relatively unchanged between time points throughout the study (p > 0.05 for all comparisons). However, Phasix™ mesh demonstrated an overall significant decrease in thickness at 24 weeks compared to 2 weeks (p < 0.05). As shown in the cross-hatched bars in Fig. 7b, the repair thickness (i.e., mesh thickness + surrounding fibrotic response) associated with Phasix™ sites was comparable between 2 and 4 weeks (p > 0.05) and then experienced minor, though statistically significant, changes between 4 and 24 weeks (p < 0.0001 for all comparisons between time points). However, the repair thickness associated with Phasix™ sites at 24 weeks ultimately remained comparable to that measured at 2 weeks (p > 0.05).

The pre-implantation thickness of Bio-A^® has been previously reported as 1.5 mm and is also depicted as a dashed line in Fig. 7a [11]. As shown in the dotted bars in Fig. 7a, the thickness of Bio-A^® was similar to its pre-implantation value at 2, 4, and 8 weeks and then began to decline sharply thereafter. In fact, a significant increase in Bio-A^® mesh thickness was observed between 2 and 4 weeks (p < 0.01), with no significant change between 4 and 8 weeks (p > 0.05), followed by significant decreases between all subsequent time points (p < 0.01 for all comparisons between 8 and 24 weeks). In addition, Bio-A^® mesh also demonstrated an overall significant decrease in mesh thickness at 24 weeks compared to 2 weeks (p < 0.0001). As shown in the gray bars of Fig. 7b, the repair thickness associated with Bio-A^® sites increased significantly between 2 and 4 weeks (p < 0.0001) and then decreased significantly between all subsequent time points (p < 0.0001 for all comparisons between 4 and 24 weeks). Additionally, the repair thickness associated with Bio-A^® sites showed an overall significant decrease at 24 weeks compared to 2 weeks (p < 0.0001).

When comparing mesh thicknesses reported for the two materials (Fig. 7a), Bio-A^® mesh exhibited significantly greater thickness than Phasix™ mesh (p < 0.05 for all comparisons) at all time points except 24 weeks when the thicknesses of the two materials were comparable (p > 0.05). In terms of the overall repair thickness (Fig. 7b), Bio-A^® sites demonstrated significantly greater repair thickness than Phasix™ sites at 2, 4, 8, and 12 weeks (p < 0.0001 in all cases). However, at 16 and 24 weeks, Phasix™ sites demonstrated significantly greater repair thickness than Bio-A^® sites (p < 0.0001 and p < 0.01, respectively).

It should be noted that inadvertent contamination of one specimen occurred that affected the Phasix™ implant on the left side of one animal. Bacterial colonization analysis, pocket swabs, and abscess scores were not recorded for this specimen and, as such, were not included in the final data analysis. Staphylococcus lentus (S. lentus) was recovered from this implant. No other implant sites had S. lentus cultured from samples collected. Confirmation plating from inoculums pre- and post-implant indicates that the contamination did not originate from the bacteria inoculum.

Fibrinous exudate (FE) and pus is primarily comprised of residual bacterial and leukocyte cellular debris and is indicative of a recent or ongoing inflammatory response. As shown in Fig. 8a, Bio-A^® demonstrated extensive FE formation on the device and within the implant pocket compared to Phasix™. As shown in Fig. 8b, at 7 days post-implantation/inoculation, Bio-A^® also demonstrated a significantly higher abscess score (median 3.0, interquartile range 1.63–3.0) representing marked abscess compared to mild abscess observed for Phasix™ (median 1.0, interquartile range 0.0–1.0, p < 0.001).