A team of researchers from Aalborg University and Aalborg University Hospital in Denmark, working with collaborators in Qatar and the United States, has now reported the first controlled in vivo evaluation of a trilayer composite electrospun scaffold for urethral reconstruction. The construct, designated CPC, consists of a core of poly(L-lactide-co-glycolide), or PLGA, sandwiched between two layers of chitosan, a naturally derived polysaccharide. The design rationale is biomimetic: the luminal chitosan layer is intended to mimic the urethral mucosa, providing a hydrophilic, bioactive surface that supports epithelial cell attachment and migration while contributing the antimicrobial properties characteristic of chitosan-based biomaterials. The central electrospun PLGA layer serves as the mechanical backbone, analogous to the submucosal tissue of the native urethral wall, offering tunable tensile strength and gradual hydrolytic degradation over the healing timeframe. The outer chitosan layer interfaces with the surrounding corpus spongiosum, the vascular tissue that envelops the urethra, promoting cellular infiltration and angiogenesis.

The biological logic behind the chitosan layers rests on well-documented immunomodulatory chemistry. Chitosan oligosaccharides act on macrophage pattern-recognition receptors to drive polarization of these immune cells toward the M2 phenotype, the anti-inflammatory state associated with tissue repair rather than with destruction. M2 macrophages secrete pro-angiogenic factors including vascular endothelial growth factor, or VEGF, supporting early vascularization of healing tissue. Chitosan additionally confers hydrophilicity and antimicrobial activity that complement the structural advantages of synthetic polyester cores. The hypothesis was that this layered architecture would attenuate the foreign-body response and support vascularization while preserving the mechanical integrity needed for surgical handling. Prior in vitro work on a related trilayer chitosan/poly(L-lactide)/chitosan construct had demonstrated superior mechanical strength, suture retention, smooth muscle cell growth, and angiogenic potential compared with monolayer PLA scaffolds, motivating the present in vivo evaluation. The Danish team substituted PLGA for PLA in the core layer because PLGA’s degradation rate can be tuned through the lactide-to-glycolide ratio, offering more flexibility than the slower-degrading PLA, and because PLGA carries broad regulatory precedent in absorbable medical devices.

What distinguishes this study from much of the preclinical urethral tissue-engineering literature is its methodological rigor. A 2021 systematic review of 28 rabbit studies published between 2014 and 2020 found a mean adherence of only 53 percent to the ARRIVE guidelines for reporting animal research, with no included study reporting sample-size estimation methodology and the majority lacking blinded outcome assessment. The new study was designed and reported in accordance with ARRIVE 2.0, directly addressing each of these documented gaps. The researchers performed an a priori sample-size calculation, prespecified a continuous quantitative primary endpoint rather than the binary outcomes such as fistula formation that have constrained formal power calculation in earlier work, and implemented blinded outcome assessment throughout. Twenty-four male New Zealand White rabbits were allocated in a one-to-one-to-one ratio to dorsal onlay urethroplasty with the trilayer CPC scaffold, with a monolayer PLGA scaffold, or to a sham surgical control, using weight-stratified allocation. Surgeons were blinded to group allocation during urethral exposure and defect creation, and because both scaffolds were thin, white, semi-flexible electrospun sheets that were not reliably distinguishable on inspection or handling, surgeons remained effectively blinded to scaffold type throughout the procedure. Retrospective misidentification of scaffold type was common when surgeons were informally asked to guess, supporting the integrity of the blinding.

The surgical model itself mirrors adult substitution urethroplasty technique. Following urethral catheterization, the penile urethra was exposed through a ventral midline skin incision, mobilized, and a standardized dorsal urethral defect measuring 10 by 3 millimeters was created. In scaffold-treated groups, a dorsal onlay graft of identical dimensions was placed on the defect and sutured with interrupted 6-0 polydioxanone monofilament sutures. To divert urine and protect the healing site, a perineostomy was created proximal to the urethroplasty, a strategy preferred over indwelling catheterization because rabbits do not reliably tolerate prolonged urethral catheters. The primary endpoint was vessel density on CD31 immunohistochemistry at 12 weeks, with secondary endpoints including tissue thickness, fibrosis, smooth muscle content, re-epithelialization, inflammatory cell scores, and urethral patency assessed by post-mortem retrograde urethrography. All histological assessments were performed by a single pathologist blinded to treatment allocation, and intra-observer reliability of vessel-density scoring was excellent, with an intraclass correlation coefficient of 0.97.

The results told a more nuanced story than the hypothesis predicted. Vessel density differed significantly between treatment groups overall, with sham animals exhibiting the highest density at a median of 78.6 vessels per square millimeter, monolayer PLGA the lowest at 39.1, and the trilayer CPC intermediate at 46.3. The direction of effect matched the in vitro pro-angiogenic activity reported for chitosan-containing constructs, and the observed CPC-PLGA median difference of 7.2 vessels per square millimeter closely matched the prespecified clinically meaningful difference of seven used in the sample-size calculation. However, the within-group variability substantially exceeded the planning assumption, reducing statistical power below the design target and widening the 95 percent confidence interval around the CPC-PLGA difference to span from minus 27.3 to 41.6 vessels per square millimeter. The trilayer did not significantly differ from the monolayer, and the overall between-group variation was driven by higher values in sham animals than in PLGA-treated animals. The authors interpret the sham finding carefully: the rabbit urethra revascularizes robustly after primary closure of a small dorsal defect, so the sham group represents an upper bound for vascularization in this model rather than a vascularization-deficient comparator.

Perhaps the most striking quantitative finding was a coherent host-response signature linking vascularization, tissue thickness, and fibrosis. Vessel density was inversely correlated with tissue thickness and with fibrosis, while tissue thickness was positively correlated with fibrosis. This pattern describes an axis of host response in which scaffold-supported remodeling produced thicker, more fibrotic, and less vascularized tissue, consistent with the behavior of synthetic scaffolds at intermediate timepoints, where a sustained foreign-body response can shift the balance from constructive vascularization toward fibrotic encapsulation. The thickness finding also carries a metric caveat: scaffold-driven regeneration produced thicker tissue at the implantation site over which a comparable absolute vessel population was distributed, biasing the per-area vessel density downward. Fibrosis itself did not differ significantly between groups, with median collagen area near 82 to 84 percent across all arms, a near-equivalence in the absence of any scaffold material suggesting that the dominant contributor to the measured collagen signal was native urethral and adjacent corpus cavernosum collagen captured within the region of interest rather than scaffold-induced scar formation.

On the inflammatory front, scaffold implantation elicited a foreign-body response in both treatment groups, with significantly elevated multinucleated giant-cell scores compared with sham. Yet the macrophage comparison provided the most defensible signal in favor of the trilayer design: median macrophage scores were lower in CPC than in PLGA, a difference that trended toward but did not reach statistical significance. This pattern is consistent with chitosan attenuating the foreign-body response, possibly through its established capacity to favor M2 over M1 macrophage polarization via chitosan-oligosaccharide signaling, while PLGA implants drive a stereotyped macrophage and giant-cell signature partly through acidic by-products of polymer hydrolysis. The authors are careful to note that this chitosan-mediated modulation is inferred from semi-quantitative macrophage counts rather than direct phenotypic markers, and that the elevated giant-cell counts in scaffold-treated groups cannot be classified as uniformly pro-inflammatory without immunohistochemistry for canonical M1 and M2 polarization markers. Meanwhile, re-epithelialization was essentially complete across all groups, with median urothelial coverage of 100 percent in the CPC group, and no animal demonstrated ulceration, unresolved wound bed, or tissue necrosis at the 12-week endpoint. The few focal regions of missing urothelium lacked the edge reaction, granulation tissue, and inflammatory infiltrate characteristic of true ulceration and were interpreted as denudation of mature urothelium, possibly a processing artifact.

Functionally, the news was uniformly good. Retrograde urethrography demonstrated preserved patency in all 23 animals completing the planned 12-week endpoint, with no fistulae or contrast extravasation observed. No animal met the prespecified stricture criterion of a 50 percent or greater reduction in urethral diameter at the implantation site, and lumen-width ratios were numerically wider in scaffold groups than in sham, a directional pattern consistent with the structural role of an onlay graft in providing a template for lumen-supporting tissue regeneration during spongiosal fibrosis. No postoperative complications or infections were observed during follow-up, and no macroscopic fistulae, adhesions, necrosis, or calculi were seen at harvest. One sham animal was euthanized early because of acute-onset bilateral hindlimb paralysis, judged unrelated to the experimental intervention, and no animals met exclusion criteria during acclimatization.

The authors conclude that the trilayer scaffold was feasible to implant and well tolerated, supporting tissue regeneration with preserved urethral patency, but that the findings refine rather than confirm the development hypothesis. They identify several priorities for future work: earlier endpoints at two and four weeks to directly characterize the chitosan-mediated angiogenic and immunomodulatory window, M1/M2 macrophage and giant-cell phenotyping to test the polarization hypothesis, mechanical-modulus tuning to match the elastic modulus of native urethral tissue at approximately 25 kilopascals, and modulation of the chitosan-to-PLGA mass ratio as a further design lever. Crosslinking strategies to slow chitosan resorption could extend the bioactive window of the surface layers, and translational evaluation will ultimately require larger animal models with urethral anatomy closer to humans, longer follow-up, and head-to-head comparison against the autologous buccal mucosa graft that remains the clinical reference standard. The rabbit dorsal onlay model itself demonstrated an excellent safety profile across all groups, supporting its utility as a reproducible platform for controlled comparison of subsequent urethral scaffolds, and the biological criteria validated here, including complete re-epithelialization without ulceration and preserved patency, are of considerable interest for hypospadias-directed scaffold development, where excessive inflammation and fibrosis contribute to fistula formation and repair failure.

Subject of Research: In vivo evaluation of a chitosan/PLGA/chitosan trilayer electrospun scaffold versus monolayer PLGA for urethral reconstruction in rabbits

Article Title: In vivo evaluation of a chitosan/PLGA/chitosan composite versus monolayer PLGA electrospun scaffold for dorsal onlay urethroplasty: a controlled study in rabbits

Article References: Mahdi, M. B., Mohtaram, F., Haase, R. N., Tuckus, G., Fojan, P., Bergman, J., Thomsen, C., Kjærgaard, B., Sørensen, S. R., Alipour, H., Abbas, T. O., Pennisi, C. P., & Nielsen, T. K. (2026). In vivo evaluation of a chitosan/PLGA/chitosan composite versus monolayer PLGA electrospun scaffold for dorsal onlay urethroplasty: a controlled study in rabbits. Frontiers in Bioengineering and Biotechnology, 14, Article 1891449. https://doi.org/10.3389/fbioe.2026.1891449

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1891449

Keywords: urethroplasty, urethral stricture, chitosan, PLGA, electrospun scaffold, tissue engineering, vascularization, foreign-body response, rabbit model, regenerative medicine, biomaterials, hypospadias