Ascorbic Acid 2-Phosphate-Releasing Supercritical Carbon Dioxide-Foamed Poly(L-Lactide-Co-epsilon-Caprolactone) Scaffolds Support Urothelial Cell Growth and Enhance Human Adipose-Derived Stromal Cell Proliferation and Collagen Production

publishedVersionPeer reviewe

Urologisten ja gynekologisten sovellusten kudosteknologiaa

Urologisten ja gynekologisten sovellusten kudosteknologiaa Kudosteknologisista hoitomuodoista on kehittymässä potentiaalinen vaihtoehto urologisiin ja gynekologisiin sovellutuksiin. Virtsaputken alahalkio (hypospadia) on yleisin peniksen synnynnäinen epämuodostuma, jossa virtsaputken kehitys on jäänyt kesken niin, että virtsaputki avautuu ventraalisesti terskan ja välilihan väliselle alueelle. Nykykäytäntö on, että hypospadia hoidetaan muotoilemalla potilaalle puuttuva osa virtsaputkea genitaalialueen kudoksista esimerkiksi esinahasta. Vakavimmissa tapauksissa voidaan käyttää genitaalialueen ulkopuolisia kudoksia, kuten posken limakalvosiirrettä, ja etenkin nämä toimenpiteet ovat erittäin herkkiä leikkauksen jälkeisille komplikaatioille kuten arpeutumiselle, ihoavanteille ja huonolle kosmeettiselle lopputulokselle. Tämän vuoksi uusien hoitomenetelmien ja vaihtoehtoisten siirremateriaalien kehittäminen on tärkeää. Tähän mennessä uroteelin kudosteknologisiin sovellutuksiin on eri tutkimuksissa testattu erilaisia luonnonperäisiä ja synteettisiä biomateriaaleja kuten soluttomia kollageenipohjaisia materiaaleja, polylaktidia ja polyglykolidia. Kuitenkaan optimaalista biomateriaalia uroteelisovellutuksiin ei ole vielä kehitetty. Uroteelisovellutuksia varten biomateriaalin pitäisi olla biohajoava, bioyhteensopiva, elastinen, helposti käsiteltävä leikkauksen yhteydessä ja sen tulisi tukea uroteelisolujen kasvua sekä virtsaputken rakennetta. Kriittistä myös on, että biomateriaali ei aiheuta haitallista tulehdus reaktiota kudoksessa. Nykyisin virtsainkontinenssin kirurgisessa hoidossa käytetään ensisijaisesti nauhaleikkauksia, jossa polypropyleeninauha asetetaan virtsaputken alle. On kuitenkin tärkeä kehittää hoitokeinoja, jotka olisivat vähemmän invasiivisia kuin kirurgiset tekniikat. Ponnistusinkontinenssin hoidossa on aiemmin käytetty myös virtsaputkea ja virtsaputken sulkijan aluetta ahtauttavia ja tiivistäviä injektiohoitoja, joissa esimerkiksi kollageeni- tai polyakryyliamidi hydrogeeliä ruiskutetaan virtsaputken alle. Ongelmana näissä hoidossa on ollut hoitotuloksen heikkeneminen ajan myötä, joten uusintainjektioita on tarvittu ylläpitämään hoitotulosta. Kudosteknologinen injektio, jossa biomateriaaligeeliä ja soluja käytetään yhdessä, voisi olla potentiaalinen tulevaisuuden hoitokeino virtsainkontinenssista kärsiville potilaille. Rasvasta eristetyt kantasolut ovat houkutteleva solulähde inkontinenssihoitoihin, sillä rasvakudosta on saatavilla runsaasti. Rasvakudoksen kantasolujen on myös todettu erilaistuvan lihassolujen suuntaan, mikä lisää niiden soveltuvuutta solulähteenä lihaskudoksen korjaamiseen ja ponnistusinkontinenssin injektiohoitoihin. Tämä väitöskirjatyö koostuu kahdesta erillisestä osiosta. Ensimmäisessä osiossa tutkittiin luonnonperäisen vesikalvon ja synteettisten polykaprolaktoni-polylaktidi komposiittikalvon sekä sileän ja teksturoidun polykaprolaktonikalvon soveltuvuutta uroteelisolujen, eli virtsateiden epiteelisolujen, kasvatusalustaksi in vitro. Työn toisessa osuudessa tutkimme soveltuvatko rasvakudoksen kantasolut yhdistettynä kollageenigeeliin virtsainkontinenssin hoitoon kliinisessä pilotti potilastutkimuksessa. Työn ensimmäisessä vaiheessa havaitsimme, että amnionkalvo ei tukenut uroteelisolujen jakautumista, elinkykyä ja fenotyyppin pysyvyyttä verrattuna polykaprolaktonikalvoon. Kaikilla tutkituilla synteettisillä kalvoilla uroteelisolut olivat elinkykyisiä ja säilyttivät fenotyyppinsä. Kuitenkin komposiitti kalvolla uroteelisolut jakautuivat hieman hitaammin kuin sileällä ja teksturoidulla polykaprolaktonikalvolla. Kliinisessä pilottitutkimuksessa totesimme, että rasvakudoksen kantasolut yhdessä kollageenin kanssa ovat turvallinen ja kohtalaisen tehokas hoitokeino käytettäväksi virtsainkontinenssin hoitoon. Lisäksi totesimme, että potilailta eristetyt rasvakudoksen kantasolut erilaistuvat lihas-, rasva-, luu- ja rustosolujen suuntaan in vitro. Yhteenvetona voidaan todeta, että tutkimusten perusteella polykaprolaktoni on potentiaalinen biomateriaali uroteelin kudosteknologisiin sovelluksiin. Lisätutkimuksia kuitenkin tarvitaan, jotta voidaan arvioida polykaprolaktonin käytettävyyttä ja bioyhteensopivuuttaa in vivo. Lisäksi myös kudosteknologiset injektiohoidot ovat lupaavia uusia menetelmiä virtsainkontinenssin hoitoon tulevaisuudessa.The tissue engineering applications has emerged as a new potential treatment method for urological and gynaecological applications. Hypospadia is the most common congenital malformation of penis, where the urethra opens ventrally to the shaft of penis or even perineum. The hypospadias are traditionally reconstructed using patients’ own genital tissue, or in severe cases non-urological graft tissue is used. However, the operations are susceptible to complications, especially in severe cases and when non-urological tissue is used. The most common complications are strictures, fistula formation and poor cosmetic outcome. Therefore, the development of alternative treatment methods is essential. Thus far, different natural and synthetic biomaterials, such as different acellular collagen based membranes, polylactide (PLA) and polyglycolide (PGA) have been studied as a growth surface for urothelial cells. However, the optimal biomaterial for urothelial applications has not been found yet. The biomaterial should be biodegradable, biocompatible, elastic, easy to handle and suture, support the urothelial cell growth and the structure of the de novo urethra. Further, the biomaterial should not evoke inflammatory tissue reaction. Nowadays, the first-line surgical treatment for urinary incontinence is the mid-urethral sling operation, though, the development of injectable bulking agents is also important in order to obtain less invasive treatments. Previously, collagen and polyacrylamide hydrogel have been used as a bulking agent to treat stress urinary incontinence. Nevertheless, the sustainability of the treatment effect has been a major problem and additional injections have been required. Therefore, tissue engineering based injection therapies using stem cells, aiming regeneration of the damaged tissue, could solve the sustainability problem. Adipose stem cells (ASCs) isolated from adipose tissue are an attractive cell source due to their abundance. Further, ASCs are known to differentiate towards myogenic cell lineages, and therefore being potential to regenerate the muscle tissue and treat urinary incontinence. This thesis is composed of two different parts. First, we studied the use of human amniotic membrane (hAM) and different synthetic biomaterial membranes, smooth (s) poly-(L-lactide-ε-co-caprolactone) (PLCL), textured (t) PLCL and knitted PLA mesh with compression moulded PLCL (cPLCL) as a growth surface for urothelial cells in vitro. In the second part, we evaluated the suitability of ASCs in combination with collagen gel to treat urinary incontinence in a clinical pilot study. In the first part, the hAM did not support the hUCs proliferation, viability and phenotype compared to the PLCL. However, on all the studied synthetic biomaterials the hUCs maintained their viability and phenotype. Further, the cPLCL supported the hUCs proliferation slightly poorer compared to the sPLCL and tPLCL. In our clinical pilot study we demonstrated that ASCs in combination with collagen gel is a safe and moderately effective treatment method for female urinary incontinence. Further, ASCs derived from the treated patients were confirmed to differentiate towards myogenic, adipogenic, osteogenic and chondrogenic cell lineages in vitro. In conclusion, PLCL membrane could be a potential biomaterial for urothelial tissue engineering. However, further research is needed to evaluate the in vivo applicability and biocompatibility of PLCL. Finally, tissue engineering based injection treatments with ASCs could be potential to treat female urinary incontinence in the future

Ascorbic Acid 2-Phosphate Releasing Supercritically Foamed Porous Poly-L-Lactide-Co-ε-Caprolactone Scaffold Enhances the Collagen Production of Human Vaginal Stromal Cells : A New Approach for Vaginal Tissue Engineering

Background:: The reconstructive surgery of vaginal defects is highly demanding and susceptible to complications, especially in larger defects requiring nonvaginal tissue grafts. Thus, tissue engineering-based solutions could provide a potential approach to the reconstruction of vaginal defects. Methods:: Here, we evaluated a novel porous ascorbic acid 2-phosphate (A2P)-releasing supercritical carbon dioxide foamed poly-L-lactide-co-ε-caprolactone (scPLCLA2P) scaffold for vaginal reconstruction with vaginal epithelial (EC) and stromal (SC) cells. The viability, proliferation, and phenotype of ECs and SCs were evaluated in monocultures and in cocultures on d 1, d 7 and d 14. Furthermore, the collagen production of SCs on scPLCLA2P was compared to that on scPLCL without A2P on d 14. Results:: Both ECs and SCs maintained their viability on the scPLCLA2P scaffold in mono- and coculture conditions, and the cells maintained their typical morphology during the 14-d culture period. Most importantly, the scPLCLA2P scaffolds supported the collagen production of SCs superior to plain scPLCL based on total collagen amount, collagen I and III gene expression results and collagen immunostaining results. Conclusion:: This is the first study evaluating the effect of A2P on vaginal tissue engineering, and the results are highly encouraging, indicating that scPLCLA2P has potential as a scaffold for vaginal tissue engineering. Graphical Abstract: [Figure not available: see fulltext.].Peer reviewe

In vitro biocompatibility of polylactide and polybutylene succinate blends for urethral tissue engineering

Surgical treatment of urothelial defects with autologous genital or extragenital tissue grafts is susceptible to complications. Tissue engineering utilizing novel biomaterials and cells such as human urothelial cells (hUC) for epithelial regeneration and adipose stromal cells (hASC) for smooth muscle restoration might offer new treatment options for urothelial defects. Previously, polylactide (PLA) has been studied for urethral tissue engineering, however, as such, it is too stiff and rigid for the application. Blending it with ductile polybutylene succinate (PBSu) could provide suitable mechanical properties for the application. Our aim was to study the morphology, viability and proliferation of hUC and hASC when cultured on 100/0 PLA/PBSu, 75/25 PLA/PBSu blend, 50/50 PLA/PBSu blend, and 0/100 PLA/PBSu discs. The results showed that the hUCs were viable and proliferated on all the studied materials. The hUCs stained pancytokeratin at 7 and 14 days, suggesting maintenance of the urothelial phenotype. The hASCs retained their viability and morphology and proliferated on all the other discs, except on PLA. On the PLA, the hASCs formed large aggregates with each other rather than attached to the material. The early smooth muscle cell markers SM22α and α-SMA were stained in hASC at 7 and 14 day time points on all PBSu-containing materials, indicating that hASCs maintain their smooth muscle differentiation potential also on PBSu. As a conclusion, PBSu is a highly potential biomaterial for urothelial tissue engineering since it supports growth and phenotypic maintenance of hUC and smooth muscle differentiation of hASC.Peer reviewe

Porous poly-L-lactide-co-epsilon-caprolactone scaffold: a novel biomaterial for vaginal tissue engineering

The surgical reconstruction of functional neovagina is challenging and susceptible to complications. Therefore, developing tissue engineering-based treatment methods for vaginal defects is important. Our aim was to develop and test a novel supercritical carbon dioxide foamed poly-L-lactide-co-1-caprolactone (scPLCL) scaffold for vaginal reconstruction. The scaffolds were manufactured and characterized for porosity (65 + 4%), pore size (350 + 150 mm) and elastic modulus (2.8 + 0.4 MPa). Vaginal epithelial (EC) and stromal cells (SC) were isolated, expanded and characterized with flow cytometry. Finally, cells were cultured with scPLCL scaffolds in separate and/or co-cultures. Their attachment, viability, proliferation and phenotype were analysed. Both cell types strongly expressed cell surface markers CD44, CD73 and CD166. Strong expression of CD326 was detected with ECs and CD90 and CD105 with SCs. Both ECs and SCs attached and maintained viability on scPLCL. Further, scPLCL supported the proliferation of especially ECs, which also maintained epithelial phenotype (cytokeratin expression) during 14-day assessment period. Interestingly, ECs expressed uroplakin (UP) Ia, UPIb and UPIII markers: further, UPIa and UPIII expression was significantly higher on ECs cultured on scPLCL than on cell culture plastic.publishedVersionPeer reviewe

Porous poly-L-lactide-co-1-caprolactone scaffold: A novel biomaterial for vaginal tissue engineering

The surgical reconstruction of functional neovagina is challenging and susceptible to complications. Therefore, developing tissue engineering-based treatment methods for vaginal defects is important. Our aim was to develop and test a novel supercritical carbon dioxide foamed poly-L-lactide-co-1-caprolactone (scPLCL) scaffold for vaginal reconstruction. The scaffolds were manufactured and characterized for porosity (65 + 4%), pore size (350 + 150 mm) and elastic modulus (2.8 + 0.4 MPa). Vaginal epithelial (EC) and stromal cells (SC) were isolated, expanded and characterized with flow cytometry. Finally, cells were cultured with scPLCL scaffolds in separate and/or co-cultures. Their attachment, viability, proliferation and phenotype were analysed. Both cell types strongly expressed cell surface markers CD44, CD73 and CD166. Strong expression of CD326 was detected with ECs and CD90 and CD105 with SCs. Both ECs and SCs attached and maintained viability on scPLCL. Further, scPLCL supported the proliferation of especially ECs, which also maintained epithelial phenotype (cytokeratin expression) during 14-day assessment period. Interestingly, ECs expressed uroplakin (UP) Ia, UPIb and UPIII markers: further, UPIa and UPIII expression was significantly higher on ECs cultured on scPLCL than on cell culture plastic.Peer reviewe

Comparison of Poly(l-lactide-co-ϵ-caprolactone) and Poly(trimethylene carbonate) Membranes for Urethral Regeneration: An in Vitro and in Vivo Study

Urethral defects are normally reconstructed using a patient's own genital tissue; however, in severe cases, additional grafts are needed. We studied the suitability of poly(l-lactide-co-ϵ-caprolactone) (PLCL) and poly(trimethylene carbonate) (PTMC) membranes for urethral reconstruction in vivo. Further, the compatibility of the materials was evaluated in vitro with human urothelial cells (hUCs). The attachment and viability of hUCs and the expression of different urothelial cell markers (cytokeratin 7, 8, 19, and uroplakin Ia, Ib, and III) were studied after in vitro cell culture on PLCL and PTMC. For the in vivo study, 32 rabbits were divided into the PLCL (n = 15), PTMC (n = 15), and control or sham surgery (n = 2) groups. An oval urethral defect 1 × 2 cm in size was surgically excised and replaced with a PLCL or a PTMC membrane or urethral mucosa in sham surgery group. The rabbits were followed for 2, 4, and 16 weeks. After the follow-up, urethrography was performed to check the patency of the urethra. The defect area was excised for histological examination, where the epithelial integrity and structure, inflammation, and fibrosis were observed. There was no notable difference on hUCs attachment on PLCL and PTMC membranes after 1 day of cell seeding, further, the majority of hUCs were viable and maintained their urothelial phenotype on both biomaterials. Postoperatively, animals recovered well, and no severe strictures were discovered by urethrography. In histological examination, the urothelial integrity and structure developed toward a normal urothelium with only mild signs of fibrosis or inflammation. According to these results, PLCL and PTMC are both suitable for reconstructing urethral defects. There were no explicit differences between the PLCL and PTMC membranes. However, PTMC membranes were more flexible, easier to suture and shape, and developed significant epithelial integrity

Comparison of Poly(l-lactide-co--caprolactone) and Poly(trimethylene carbonate) Membranes for Urethral Regeneration:An In Vitro and In Vivo Study

Urethral defects are normally reconstructed using a patient's own genital tissue; however, in severe cases, additional grafts are needed. We studied the suitability of poly(l-lactide-co--caprolactone) (PLCL) and poly(trimethylene carbonate) (PTMC) membranes for urethral reconstruction in vivo. Further, the compatibility of the materials was evaluated in vitro with human urothelial cells (hUCs). The attachment and viability of hUCs and the expression of different urothelial cell markers (cytokeratin 7, 8, 19, and uroplakin Ia, Ib, and III) were studied after in vitro cell culture on PLCL and PTMC. For the in vivo study, 32 rabbits were divided into the PLCL (n=15), PTMC (n=15), and control or sham surgery (n=2) groups. An oval urethral defect 1x2cm in size was surgically excised and replaced with a PLCL or a PTMC membrane or urethral mucosa in sham surgery group. The rabbits were followed for 2, 4, and 16 weeks. After the follow-up, urethrography was performed to check the patency of the urethra. The defect area was excised for histological examination, where the epithelial integrity and structure, inflammation, and fibrosis were observed. There was no notable difference on hUCs attachment on PLCL and PTMC membranes after 1 day of cell seeding, further, the majority of hUCs were viable and maintained their urothelial phenotype on both biomaterials. Postoperatively, animals recovered well, and no severe strictures were discovered by urethrography. In histological examination, the urothelial integrity and structure developed toward a normal urothelium with only mild signs of fibrosis or inflammation. According to these results, PLCL and PTMC are both suitable for reconstructing urethral defects. There were no explicit differences between the PLCL and PTMC membranes. However, PTMC membranes were more flexible, easier to suture and shape, and developed significant epithelial integrity