Engineering vascularized soft tissue

Pehmytkudos käsittää elimiä yhdistävät, tukevat ja ympäröivät pehmeät kudokset kuten sidekudoksen, lihakset, hermot, verisuonet ja rasvakudoksen. Rasvakudoksen on todettu olevan sekä aktiivinen kudos että erinomainen ja helposti saatavilla oleva kasvutekijöiden ja kantasolujen lähde. Pehmytkudosvaurioista kärsiviä potilaita ja kroonisten haavojen tai arpikudosten hoitoa tarvitsevia potilaita on lukuisa joukko, eikä heille ole tällä hetkellä tarjolla parantavaa hoitomuotoa. Kudosteknologia pyrkii tuottamaan uusia kudosrakenteita laboratoriossa, mutta ongelmana toimivien kudossiirrännäisten kehittämisessä on riittävän verisuonituksen puuttuminen, mikä merkittävästi rajoittaa uudentyyppisten kudosteknologisten hoitomuotojen kehittämistä. Verisuonituksen muodostumisen ja sen estämisen tutkiminen on lisäksi tärkeää useissa muissa sairauksissa kuten syövissä. Väitöskirjassa tutkittiin verisuonituksen ja pehmytkudoksen kehittämistä kudosteknologisin keinoin. Työssä kehitettiin lisäksi solutestimenetelmiä verisuonimuodostuksen tutkimiseen. Ensimmäisessä ja toisessa osatyössä tutkittiin ihmisen rasvakudoksesta eristetyn soluttoman proteiini- ja kasvutekijäuutteen kykyä aikaansaada uutta pehmytkudosta. Rasvakudosuute lisäsi solumalleissa sekä verisuonirakenteiden muodostumista että rasvakudoksen kantasolujen erilaistumista rasvasoluiksi. Rasvakudosuutetta sisältävän siirrännäisen todettiin aikaansaavan verisuonituksen muodostumista ja kypsän uuden rasvakudoksen syntymistä ihonalaiskudoksessa. Materiaalin todettiin olevan kudosyhteensopiva. Työssä tutkittu rasvakudosuute on lupaava soluton hoitomuoto käytettäväksi verisuonimuodostuksen ja pehmytkudoksen aikaansaamiseksi pehmytkudosvaurioissa. Kolmannessa osatyössä kehitettiin rasvakudoksen kantasoluihin perustuva kolmiulotteinen verisuonimalli, joka muistuttaa ihmiskudoksen verisuonirakennetta. Tätä solumallia voidaan käyttää verisuonitutkimuksessa sekä kehitettäessä edistyneempiä ihmiskudosmalleja. Malli on sovellettavissa myös hoitotarkoituksiin tehtäviin kudossiirrännäisiin. Neljännessä osatyössä tutkimustasoisesta verisuonisolumallista kehitettiin kansainvälisten standardien mukaisesti toistettava solutestimenetelmä. Testiä voidaan käyttää luotettavana menetelmänä lääkeaineiden ja kemikaalien turvallisuuden ja tehon testauksessa.Tissue engineering aims at replacing or regenerating tissue structures. In tissue engineering, the most limiting factor is the successful vascularization of the transplanted construct or the transplantation area. The current major hurdle is the lack of a therapeutic method that would rapidly induce adequate vascularization which would also remain in tissue. The proper method for induction of vascularization would help several tissue engineering applications as well as numerous patients suffering from ischemic diseases, chronic wounds and soft tissue defects. In addition to the tissue engineering applications, investigation of the angiogenesis process is important for the treatment of different other diseases such as cancers. Therefore, in vitro angiogenesis assays that can predict human effects are required. Understanding the role of angiogenesis in adipose tissue is of especial importance, as vasculature regulates both the adipose tissue mass development and adipose tissue reduction and obesity is a cause of a distruction in normal adipose tissue homeostasis. The aim of the current study was to study angiogenesis and adipogenesis induction in in vitro assays and in vivo. In the first part of the study, acellular angiogenic and adipogenic agent was extracted from adipose tissue and the bioactivity of the extract was tested in vitro and in vivo. In cell culture studies, this adipose tissue extract was shown to stimulate angiogenesis and adipose tissue stromal cell maturation towards adipocytes. In vivo, when combined with hyaluronan hydrogel, the extract was shown to induce sustained soft tissue formation. No hypersensitivity or foreign body reactions were seen. Adipose tissue extract has therefore potential to be used as an acellular alternative in the treatment of soft tissue defects for reintroducing soft tissue at the defect sites. Adipose tissue extract has also potential to be used to induce revascularization in ischemic tissues and to be used in other tissue engineering products that fail due to inadequate vascularization. Moreover, adipose tissue extract can be used as an inducer in an in vitro model of natural adipogenesis. The second part of the study focused on developing in vitro methods for angiogenesis induction. We created a multilayered adipose stromal cell vascular network with properties of maturating vessels that can be used for studying angiogenesis in vitro, and especially, in the development of in vitro three dimensional tissue models as well as possibly as a vascularized platform in implantable soft tissue constructs. We also intra-laboratory validated an in vitro angiogenesis assay to be used as a routine cell assay for drug and chemical screening. The currently validated in vitro assay is a relevant-to-human bioassay that can be used for preclinical drug efficacy screening studies, and in addition, is applicable also for testing angiotoxicity of chemicals

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K+ depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E2 treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells’ population growth by inducing maturation and differentiation

GENE EXPRESSION PROFILING AFTER ANGIOGENESIS INHIBITOR TREATMENT

The angiogenic process can be summarized as cell activation by a lack of oxygen releases angiogenic molecules that attract inflammatory and endothelial cells and promote their proliferation. Several protein fragments produced by the digestion of the blood-vessel walls intensify the proliferative activity of endothelial cells. Acetyl salicylic acid is often used as an analgesic drug to relieve minor aches and pains, a drug with antitumour activity and an anti-inflammatory medication. In our experiments we propose using the angiogenesis model and photodynamic therapy (PDT) for observing changes in angiogenesis after treatment, and also for increasing the effect of PDT by addition of angiogenesis inhibitors

Intra-Laboratory Pre-Validation of a Human Cell Based in vitro Angiogenesis Assay for Testing Angiogenesis Modulators

The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory pre-validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis, e.g., pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using six reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory pre-validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation), batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability) were tested. The pre-set acceptance criteria for the intra-laboratory pre-validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests

THE EFFECT OF ACETYLSALICYLIC ACID ON ANGIOGENESIS IN VITRO

Angiogenesis, the formation of new blood vessels, is an essential aspect of, among others, embryonic development, wound healing and the female reproductive cycle. It is also necessary for the expansion of tumour masses beyond a minute volume. Acetylsalicylic acid (ASA) is a non-steroidal anti-inflammatory drug with additional antitumour activity. We tested ASA for its ability to inhibit angiogenesis in a simplified angiogenesis model, hASC+HUVEC co cultured in vitro, using immunocytochemical staining with fluorescence-marked antibodies and observation of tubulelike structures and their branching under a fluorescence microscope. We confirmed that ASA is an efficient and useful angiogenesis inhibitor and deserves further attention. We intend using the designed angiogenesis model and the methods described for observing changes in angiogenesis after anti tumour photodynamic therapy (PDT), and also for enhancing PDT efficiency by addition of angiogenesis inhibitors

The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells from human cord blood

Background aims. Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. Methods. CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. Results. We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. Conclusions. Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.Peer reviewe

Engineering vascularized soft tissue

Pehmytkudos käsittää elimiä yhdistävät, tukevat ja ympäröivät pehmeät kudokset kuten sidekudoksen, lihakset, hermot, verisuonet ja rasvakudoksen. Rasvakudoksen on todettu olevan sekä aktiivinen kudos että erinomainen ja helposti saatavilla oleva kasvutekijöiden ja kantasolujen lähde. Pehmytkudosvaurioista kärsiviä potilaita ja kroonisten haavojen tai arpikudosten hoitoa tarvitsevia potilaita on lukuisa joukko, eikä heille ole tällä hetkellä tarjolla parantavaa hoitomuotoa. Kudosteknologia pyrkii tuottamaan uusia kudosrakenteita laboratoriossa, mutta ongelmana toimivien kudossiirrännäisten kehittämisessä on riittävän verisuonituksen puuttuminen, mikä merkittävästi rajoittaa uudentyyppisten kudosteknologisten hoitomuotojen kehittämistä. Verisuonituksen muodostumisen ja sen estämisen tutkiminen on lisäksi tärkeää useissa muissa sairauksissa kuten syövissä. Väitöskirjassa tutkittiin verisuonituksen ja pehmytkudoksen kehittämistä kudosteknologisin keinoin. Työssä kehitettiin lisäksi solutestimenetelmiä verisuonimuodostuksen tutkimiseen. Ensimmäisessä ja toisessa osatyössä tutkittiin ihmisen rasvakudoksesta eristetyn soluttoman proteiini- ja kasvutekijäuutteen kykyä aikaansaada uutta pehmytkudosta. Rasvakudosuute lisäsi solumalleissa sekä verisuonirakenteiden muodostumista että rasvakudoksen kantasolujen erilaistumista rasvasoluiksi. Rasvakudosuutetta sisältävän siirrännäisen todettiin aikaansaavan verisuonituksen muodostumista ja kypsän uuden rasvakudoksen syntymistä ihonalaiskudoksessa. Materiaalin todettiin olevan kudosyhteensopiva. Työssä tutkittu rasvakudosuute on lupaava soluton hoitomuoto käytettäväksi verisuonimuodostuksen ja pehmytkudoksen aikaansaamiseksi pehmytkudosvaurioissa. Kolmannessa osatyössä kehitettiin rasvakudoksen kantasoluihin perustuva kolmiulotteinen verisuonimalli, joka muistuttaa ihmiskudoksen verisuonirakennetta. Tätä solumallia voidaan käyttää verisuonitutkimuksessa sekä kehitettäessä edistyneempiä ihmiskudosmalleja. Malli on sovellettavissa myös hoitotarkoituksiin tehtäviin kudossiirrännäisiin. Neljännessä osatyössä tutkimustasoisesta verisuonisolumallista kehitettiin kansainvälisten standardien mukaisesti toistettava solutestimenetelmä. Testiä voidaan käyttää luotettavana menetelmänä lääkeaineiden ja kemikaalien turvallisuuden ja tehon testauksessa.Tissue engineering aims at replacing or regenerating tissue structures. In tissue engineering, the most limiting factor is the successful vascularization of the transplanted construct or the transplantation area. The current major hurdle is the lack of a therapeutic method that would rapidly induce adequate vascularization which would also remain in tissue. The proper method for induction of vascularization would help several tissue engineering applications as well as numerous patients suffering from ischemic diseases, chronic wounds and soft tissue defects. In addition to the tissue engineering applications, investigation of the angiogenesis process is important for the treatment of different other diseases such as cancers. Therefore, in vitro angiogenesis assays that can predict human effects are required. Understanding the role of angiogenesis in adipose tissue is of especial importance, as vasculature regulates both the adipose tissue mass development and adipose tissue reduction and obesity is a cause of a distruction in normal adipose tissue homeostasis. The aim of the current study was to study angiogenesis and adipogenesis induction in in vitro assays and in vivo. In the first part of the study, acellular angiogenic and adipogenic agent was extracted from adipose tissue and the bioactivity of the extract was tested in vitro and in vivo. In cell culture studies, this adipose tissue extract was shown to stimulate angiogenesis and adipose tissue stromal cell maturation towards adipocytes. In vivo, when combined with hyaluronan hydrogel, the extract was shown to induce sustained soft tissue formation. No hypersensitivity or foreign body reactions were seen. Adipose tissue extract has therefore potential to be used as an acellular alternative in the treatment of soft tissue defects for reintroducing soft tissue at the defect sites. Adipose tissue extract has also potential to be used to induce revascularization in ischemic tissues and to be used in other tissue engineering products that fail due to inadequate vascularization. Moreover, adipose tissue extract can be used as an inducer in an in vitro model of natural adipogenesis. The second part of the study focused on developing in vitro methods for angiogenesis induction. We created a multilayered adipose stromal cell vascular network with properties of maturating vessels that can be used for studying angiogenesis in vitro, and especially, in the development of in vitro three dimensional tissue models as well as possibly as a vascularized platform in implantable soft tissue constructs. We also intra-laboratory validated an in vitro angiogenesis assay to be used as a routine cell assay for drug and chemical screening. The currently validated in vitro assay is a relevant-to-human bioassay that can be used for preclinical drug efficacy screening studies, and in addition, is applicable also for testing angiotoxicity of chemicals