Tissue response and wound healing after placement of two types of bioengineered grafts containing vital cells in submucosal maxillary pouches: an experimental pilot study in rabbits

PURPOSE: This pilot study evaluated the wound healing and tissue response after placement of two different skin substitutes in subgingival mucosal pouches in rabbits. MATERIALS AND METHODS: Four rabbits were selected to receive a commercially available skin substitute consisting of a collagen matrix with fibroblasts and an epithelial layer (test membrane 1) and a prototype device consisting of a collagen matrix with fibroblasts only (test membrane 2). In each rabbit, two horizontal incisions were made in the buccal alveolar mucosa of the maxilla bilaterally to create submucosal pouches. Three pouches in each animal were filled with either the test 1 or test 2 membranes, and one pouch was left without a membrane (sham-operated control). All rabbits were sacrificed after a healing period of 4 weeks, and histologic samples were prepared and examined. RESULTS: After a healing period of 1 month, both tested membranes were still visible in the sections. Test membrane 1 was still bilayered, contained inflammatory cells in its center, and was encapsulated by a thick fibrous tissue. Numerous ectopic calcifications were evident in the collagenous part of the membrane and in association with some basal epithelial cells. Test membrane 2 was also encapsulated in fibrous tissue, with inflammatory cells present only between the fibrous encapsulation and the remnants of the membrane. For test membrane 2, no calcifications were visible. CONCLUSIONS: Test membrane 1 seemed to be more resistant to degradation, but there was also a more pronounced inflammatory reaction in comparison to test membrane 2, especially in the vicinity of the keratinocytes. The significance of the ectopic calcifications, along with that of the resorption or degradation processes of both tested membranes, must be evaluated in future experimental studies, with different time points after implantation examine

Bioengineered periodontal tissue formed on titanium dental implants

The ability to use autologous dental progenitor cells (DPCs) to form organized periodontal tissues on titanium implants would be a significant improvement over current implant therapies. Based on prior experimental results, we hypothesized that rat periodontal ligament (PDL)-derived DPCs can be used to bioengineer PDL tissues on titanium implants in a novel, in vivo rat maxillary molar implant model. Analyses of recovered implants revealed organized PDL tissues surrounding titanium implant surfaces in PDL-cell-seeded, and not in unseeded control, implants. Rat PDL DPCs also exhibited differentiative potential characteristic of stem cells. These proof-of-principle findings suggest that PDL DPCs can organize periodontal tissues in the jaw, at the site of previously lost teeth, indicating that this method holds potential as an alternative approach to osseointegrated dental implants. Further refinement of this approach will facilitate the development of clinically relevant methods for autologous PDL regeneration on titanium implants in humans.

Computer-assisted arthroplasty using bioengineered autografts

Recent advances in tissue-engineered cartilage open the door to new clinical treatments of joint lesions. Common to all therapies with in-vitro-engineered autografts is the need for optimal fit of the construct to allow screwless implantation and optimal integration into the live joint. Computer-assisted surgery (CAS) techniques are prime candidates to ensure the required accuracy, while at the same time simplifying the procedure. A pilot study has been conducted aiming at assembling a new set of methods to support ankle joint arthroplasty using bioengineered autografts. Computer assistance allows planning of the implant shape on a computed tomography (CT) image, manufacturing the construct according to the plan, and interoperatively navigating the surgical tools for implantation. A rotational symmetric model of the joint surface was used to avoid segmentation of the CT image; new software was developed to determine the joint axis and make the implant shape parameterizable. A complete cycle of treatment from planning to operation was conducted on a human cadaveric foot, thus proving the feasibility of computer-assisted arthroplasty using bioengineered autografts

Enhancement of titanium implants via bioengineered periodontal tissues

Osseointegration of titanium dental implants into the jaw bone, which is required for maintenance of the implant in the jaw, results in ankylosis. Dental implants are therefore very unlike natural teeth, which exhibit significant movement in response to mechanical forces. The ability to generate periodontal ligament (PDL) tissues onto dental implants would better mimic the functional characteristics of natural teeth, and would likely improve implant duration and function. OBJECTIVES: The objective of this study was to investigate the feasibility of bioengineering PDL tissues onto titanium implant surfaces. METHODS: Bilateral maxillary first and second molars of 8-week old rats were extracted and used to generate single cell suspensions of PDL tissues, which were expanded in culture. Immunohistochemistry and RT-PCR were used to identify putative PDL progenitor/stem cell populations and characterize stem cell properties, including self-renewal, multipotency and stem cell maker expression. Cultured rPDL cells were harvested at third passage, seeded onto Matrigel-coated titanium implants (1.75 mm x 1 mm), and placed into healed M1/M2 extraction sites. Non-cell seeded Matrigel-coated titanium implants served as negative controls. Implants were harvested after 8, 12, or 18 weeks. RESULTS: Cultured rPDL cells expressed the mesenchymal stem-cell marker STRO-1. Under defined culture conditions, PDL cells differentiated into adipogenic, neurogenic and osteogenic lineages. While control implants were largely surrounded by alveolar bone, experimental samples exhibited fibrous PDL-like tissues, and perhaps cementum, on the surface of experimental implants. CONCLUSIONS: PDL contains stem cells that can generate cementum/PDL-like tissue in vivo. Transplantation of these cells might hold promise as a therapeutic approach for the bioengineering of PDL tissues onto titanium implant. Further refinement of this method will likely result in improved dental implant strategies for use of autologous PDL tissue regeneration in humans. This research was supported by CIMIT, and NIH/NIDCR grant DE016132 (PCY), and TEACRS (YL).

Aggravation of cardiac myofibroblast arrhythmogeneicity by mechanical stress

Aims Myofibroblasts (MFBs) as appearing in the myocardium during fibrotic remodelling induce slow conduction following heterocellular gap junctional coupling with cardiomyocytes (CMCs) in bioengineered tissue preparations kept under isometric conditions. In this study, we investigated the hypothesis that strain as developed during diastolic filling of the heart chambers may modulate MFB-dependent slow conduction. Methods and results Effects of defined levels of strain on single-cell electrophysiology (patch clamp) and impulse conduction in patterned growth cell strands (optical mapping) were investigated in neonatal rat ventricular cell cultures (Wistar) grown on flexible substrates. While 10.5% strain only minimally affected conduction times in control CMC strands (+3.2%, n.s.), it caused a significant slowing of conduction in the fibrosis model consisting of CMC strands coated with MFBs (conduction times +26.3%). Increased sensitivity to strain of the fibrosis model was due to activation of mechanosensitive channels (MSCs) in both CMCs and MFBs that aggravated the MFB-dependent baseline depolarization of CMCs. As found in non-strained preparations, baseline depolarization of CMCs was partly due to the presence of constitutively active MSCs in coupled MFBs. Constitutive activity of MSCs was not dependent on the contractile state of MFBs, because neither stimulation (thrombin) nor suppression (blebbistatin) thereof significantly affected conduction velocities in the non-strained fibrosis model. Conclusions The findings demonstrate that both constitutive and strain-induced activity of MSCs in MFBs significantly enhance their depolarizing effect on electrotonically coupled CMCs. Ensuing aggravation of slow conduction may contribute to the precipitation of strain-related arrhythmias in fibrotically remodelled hearts.

Tackling feline infectious peritonitis via reverse genetics

Feline infectious peritonitis (FIP) is caused by feline coronaviruses (FCoVs) and represents one of the most important lethal infectious diseases of cats. To date, there is no efficacious prevention and treatment, and our limited knowledge on FIP pathogenesis is mainly based on analysis of experiments with field isolates. In a recent study, we reported a promising approach to study FIP pathogenesis using reverse genetics. We generated a set of recombinant FCoVs and investigated their pathogenicity in vivo. The set included the type I FCoV strain Black, a type I FCoV strain Black with restored accessory gene 7b, two chimeric type I/type II FCoVs and the highly pathogenic type II FCoV strain 79-1146. All recombinant FCoVs and the reference strain isolates were found to establish productive infections in cats. While none of the type I FCoVs and chimeric FCoVs induced FIP, the recombinant type II FCoV strain 79-1146 was as pathogenic as the parental isolate. Interestingly, an intact ORF 3c was confirmed to be restored in all viruses (re)isolated from FIP-diseased animals.