Methacrylated gellan gum hydrogels, obtained either by ionic- (iGGMA)
and photo-crosslinking (phGG-MA), have been investigated as
potential biomaterials for supporting nucleus pulposus (NP) regeneration
and/or repair [1,2]. In previous work, some advantages were
attributed to GG-MA hydrogels, such as: (i) the possibility to control
endothelial cells infiltration and blood vessel ingrowth’s, (ii) tunable
and improved mechanical properties, and (iii) in situ gelation, within
seconds to few minutes. In this study, the mechanical and biological
performance of these hydrogels was firstly evaluated in vitro. Human
intervertebral disc (hIVD) cells obtained from herniated patients were
cultured within both hydrogels, for 1 up to 21 days. Dynamic mechanical
analysis and biological characterization (calcein-AM staining, ATP
and DNA quantification and PCR) were performed after specific times
of culturing. A biocompatibility study was also performed in vivo, by
subcutaneous implantation of acellular iGG-MA and phGG-MA hydrogels
in Lewis rats for the period of 10 and 18 days. Tissue response to
the hydrogels implantation was determined by histological analysis
(haematoxylin-eosin staining). The in vitro study showed that both cell
loading and culturing time do not have an effect on the mechanical
properties of the hydrogels. Regarding their biological performance,
the iGG-MA and phGG-MA hydrogels showed to be effective on supporting
hIVD cells encapsulation and viability up to 21 days of culturing.
Human IVD cells were homogeneously distributed within the
hydrogels and maintained its round-shape morphology during culturing
time. The in vivo biocompatibility study showed that iGG-MA and
phGG-MA hydrogels do not elicit any deleterious effect, as denoted by
the absence of necrosis and calcification, or acute inflammatory reaction.
A thin fibrous capsule was observed around the implanted hydrogels.
The results presented in this study indicate that the iGG-MA and
phGG-MA hydrogels are stable in vitro and in vivo, support hIVD cells
encapsulation and viability, and were found to be well-tolerated and
non-cytotoxic in vivo, thus being potential candidates for NP regeneration

Abatangelo, G.

Mano, J. F.

Mendes, João Espregueira

Oliveira, Joaquim M.

Oliveira, Mariana B.

Pereira, H.

Reis, R. L.

Silva-Correia, Joana

Vindigni, V.

Zavan, B.

Journal of Tissue Engineering and Regenerative Medicine

Universidade do Minho: RepositoriUM

TS021Mechanical performance and biocompatibilitystudy of methacrylated Gellan gum hydrogelswith potential for nucleus pulposus regenerationJ Silva-Correia1,2, B Zavan3, V Vindigni4, MB Oliveira1,2,JF Mano1,2, H Pereira1,2,5,6, J M Oliveira1,2,JD Espregueira-Mendes1,2,5, G Abatangelo3 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Guimara˜es, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimara˜es, Portugal;3Department of Histology, Microbiology and BiomedicalTechnology, Univ. Padova, Viale G. Colombo 3, Padova, Italy;4Department of Plastic Surgery, Univ. Padova, Via Giustiniani44, Padova, Italy; 5Sau´de Atlaˆntica Sports Center – F.C. PortoStadium, Minho University and Porto University Research Center,Portugal; 6Orthopedic Department Centro Hospitalar Po´voa deVarzim – Vila do Conde, PortugalMethacrylated gellan gum hydrogels, obtained either by ionic- (iGG-MA) and photo-crosslinking (phGG-MA), have been investigated aspotential biomaterials for supporting nucleus pulposus (NP) regenera-tion and/or repair [1,2]. In previous work, some advantages wereattributed to GG-MA hydrogels, such as: (i) the possibility to controlendothelial cells infiltration and blood vessel ingrowth’s, (ii) tunableand improved mechanical properties, and (iii) in situ gelation, withinseconds to few minutes. In this study, the mechanical and biologicalperformance of these hydrogels was firstly evaluated in vitro. Humanintervertebral disc (hIVD) cells obtained from herniated patients werecultured within both hydrogels, for 1 up to 21 days. Dynamic mechani-cal analysis and biological characterization (calcein-AM staining, ATPand DNA quantification and PCR) were performed after specific timesof culturing. A biocompatibility study was also performed in vivo, bysubcutaneous implantation of acellular iGG-MA and phGG-MA hydro-gels in Lewis rats for the period of 10 and 18 days. Tissue response tothe hydrogels implantation was determined by histological analysis(haematoxylin-eosin staining). The in vitro study showed that both cellloading and culturing time do not have an effect on the mechanicalproperties of the hydrogels. Regarding their biological performance,the iGG-MA and phGG-MA hydrogels showed to be effective on sup-porting hIVD cells encapsulation and viability up to 21 days of cultur-ing. Human IVD cells were homogeneously distributed within thehydrogels and maintained its round-shape morphology during cultur-ing time. The in vivo biocompatibility study showed that iGG-MA andphGG-MA hydrogels do not elicit any deleterious effect, as denoted bythe absence of necrosis and calcification, or acute inflammatory reac-tion. A thin fibrous capsule was observed around the implanted hydro-gels. The results presented in this study indicate that the iGG-MA andphGG-MA hydrogels are stable in vitro and in vivo, support hIVD cellsencapsulation and viability, and were found to be well-tolerated andnon-cytotoxic in vivo, thus being potential candidates for NP regenera-tion.Acknowledgements: Funding from EU FP7 project Disc Regeneration(NMP3-LA-2008-213904).References:1. J. Silva-Correia, et al., J Tissue Eng Regen Med, 2011, 5(6):e97-e107.2. J. Silva-Correia, et al., Tissue Eng Part A, 2012, 18(11–12):1203–1212.TS022Advanced mimetic materials for meniscus tissueengineering: targeting segmental vascularizationJ Silva-Correia1,2, H Pereira1,2,3,4, L-P Yan1,2, V Miranda-Gonc¸alves2,5, AL Oliveira1,2, JM Oliveira1,2, RM Reis2,5,6,JD Espregueira-Mendes1,2,3 and RL Reis1,213B’s Research Group - Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Guimara˜es, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimara˜es, Portugal;3Sau´de Atlaˆntica Sports Center – F.C. Porto Stadium, MinhoUniversity and Porto University Research Center, Portugal;4Orthopedic Department Centro Hospitalar Po´voa de Varzim –Vila do Conde, Portugal; 5Life and Health Science ResearchInstitute (ICVS), School of Health Sciences, University of Minho,Campus de Gualtar, Braga, Portugal; 6Molecular OncologyResearch Center, Barretos Cancer Hospital, Barretos, Sa˜o Paulo,BrazilMeniscus lesions are among the most common orthopaedic injurieswhich can ultimately lead to degeneration of the knee articular carti-lage. The human meniscus has a limited healing potential, partly dueto a poor vasculature, and thus meniscus regeneration using tissueengineering strategies has recently been investigated as a promisingalternative to total/partial meniscectomy [1]. Advanced scaffolds fortissue engineering of meniscus should be able to mimic and preservethe asymmetric vascular network of this complex tissue, i.e. enable con-trolling the segmental vascularization during the regeneration process.Novel scaffolds were produced combining a silk polymeric matrix (12wt%) [2] and the methacrylated gellan gum hydrogel (iGG-MA),which has been shown to be able to prevent the ingrowth of endothe-lial cells and blood vessels into the hydrogels [3,4]. The angiogenic/anti-angiogenic potential of acellular and cell-laden silk-12 scaffoldscombined with iGG-MA hydrogel was investigated in vivo, using thechick embryo chorioallantoic membrane (CAM) assay. For producingthe cell-laden scaffolds, human meniscus cells (HMC¢s) were isolatedfrom morphologically intact human menisci using an enzymatic-baseddigestion and expanded using standard culture conditions. The HMC’s-laden hydrogel/silk scaffolds were produced by encapsulating theHMC’s into a 2 wt% GG-MA hydrogel, followed by impregnation ontothe 12 wt% silk scaffold and ionic-crosslinking in a saline solution. ACAM assay was used to investigate the control of segmental vasculari-zation of the acellular and HMC¢s-laden hydrogel/silk scaffolds by theeffect of GG-MA hydrogel, until day 14 of embryonic development.The in vivo study allowed investigating the number of macroscopicblood vessels converging to the implants. The evaluation of possibleinflammation and endothelial cells ingrowths was performed by histo-logical (haematoxylin and eosin - H&E - staining) and immunohisto-chemical methods (SNA-lectin staining). When the silk-12 scaffold wascombined with the hydrogel, an inhibitory effect was observed as dem-onstrated by the low number of convergent blood vessels. Results haveshown that iGG-MA hydrogel prevented the endothelial cells adhesionand blood vessels infiltration into the HMC’s hydrogel/silk scaffolds,after 4 days of implantation. This study showed that the hydrogel/silkscaffolds enabled controlling the segmental vascularization, thus it canpossibly mimic the native vasculature architecture during meniscusregeneration.References:1. H. Pereira, et al., Arthroscopy, 2011, 27(12):1706–1719.2. L.-P. Yan, et al., Acta Biomater, 2012, 8(1):289–301.3. J. Silva-Correia, et al., J Tissue Eng Regen Med, 2011, 5(6):e97-e107.4. J. Silva-Correia, et al., Tissue Eng Part A, 2012, 18(11–12):1203–1212. 2012 The Authors J Tissue Eng Regen Med 2012; 6 (Suppl. 2): 8–39.Journal of Tissue Engineering and Regenerative Medicine 2012 John Wiley & Sons, Ltd. DOI: 10.1002/term.160818 Abstracts List

Mechanical performance and biocompatibility study of methacrylated Gellan gum hydrogels with potential for nucleus pulposus regeneration

TS021
Mechanical performance and biocompatibility
study of methacrylated Gellan gum hydrogels
with potential for nucleus pulposus regeneration
J Silva-Correia1,2, B Zavan3, V Vindigni4, MB Oliveira1,2,
JF Mano1,2, H Pereira1,2,5,6, J M Oliveira1,2,
JD Espregueira-Mendes1,2,5, G Abatangelo3 and RL Reis1,2
13B’s Research Group – Biomaterials, Biodegradables and
Biomimetics, University of Minho, Headquarters of the European
Institute of Excellence on Tissue Engineering and Regenerative
Medicine, AvePark, Guimarães, Portugal; 2ICVS/3B’s - PT
Government Associate Laboratory, Braga/Guimarães, Portugal;
3Department of Histology, Microbiology and Biomedical
Technology, Univ. Padova, Viale G. Colombo 3, Padova, Italy;
4Department of Plastic Surgery, Univ. Padova, Via Giustiniani
44, Padova, Italy; 5Saúde Atlântica Sports Center – F.C. Porto
Stadium, Minho University and Porto University Research Center,
Portugal; 6Orthopedic Department Centro Hospitalar Póvoa de
Varzim – Vila do Conde, Portugal
Methacrylated gellan gum hydrogels, obtained either by ionic- (iGG-
MA) and photo-crosslinking (phGG-MA), have been investigated as
potential biomaterials for supporting nucleus pulposus (NP) regenera-
tion and/or repair [1,2]. In previous work, some advantages were
attributed to GG-MA hydrogels, such as: (i) the possibility to control
endothelial cells infiltration and blood vessel ingrowth’s, (ii) tunable
and improved mechanical properties, and (iii) in situ gelation, within
seconds to few minutes. In this study, the mechanical and biological
performance of these hydrogels was firstly evaluated in vitro. Human
intervertebral disc (hIVD) cells obtained from herniated patients were
cultured within both hydrogels, for 1 up to 21 days. Dynamic mechani-
cal analysis and biological characterization (calcein-AM staining, ATP
and DNA quantification and PCR) were performed after specific times
of culturing. A biocompatibility study was also performed in vivo, by
subcutaneous implantation of acellular iGG-MA and phGG-MA hydro-
gels in Lewis rats for the period of 10 and 18 days. Tissue response to
the hydrogels implantation was determined by histological analysis
(haematoxylin-eosin staining). The in vitro study showed that both cell
loading and culturing time do not have an effect on the mechanical
properties of the hydrogels. Regarding their biological performance,
the iGG-MA and phGG-MA hydrogels showed to be effective on sup-
porting hIVD cells encapsulation and viability up to 21 days of cultur-
ing. Human IVD cells were homogeneously distributed within the
hydrogels and maintained its round-shape morphology during cultur-
ing time. The in vivo biocompatibility study showed that iGG-MA and
phGG-MA hydrogels do not elicit any deleterious effect, as denoted by
the absence of necrosis and calcification, or acute inflammatory reac-
tion. A thin fibrous capsule was observed around the implanted hydro-
gels. The results presented in this study indicate that the iGG-MA and
phGG-MA hydrogels are stable in vitro and in vivo, support hIVD cells
encapsulation and viability, and were found to be well-tolerated and
non-cytotoxic in vivo, thus being potential candidates for NP regenera-
tion.
Acknowledgements: Funding from EU FP7 project Disc Regeneration
(NMP3-LA-2008-213904).
References:
1. J. Silva-Correia, et al., J Tissue Eng Regen Med, 2011, 5(6):e97-
e107.
2. J. Silva-Correia, et al., Tissue Eng Part A, 2012, 18(11–12):1203–
1212.
TS022
Advanced mimetic materials for meniscus tissue
engineering: targeting segmental vascularization
J Silva-Correia1,2, H Pereira1,2,3,4, L-P Yan1,2, V Miranda-
Gonçalves2,5, AL Oliveira1,2, JM Oliveira1,2, RM Reis2,5,6,
JD Espregueira-Mendes1,2,3 and RL Reis1,2
13B’s Research Group - Biomaterials, Biodegradables and
Biomimetics, University of Minho, Headquarters of the European
Institute of Excellence on Tissue Engineering and Regenerative
Medicine, AvePark, Guimarães, Portugal; 2ICVS/3B’s - PT
Government Associate Laboratory, Braga/Guimarães, Portugal;
3Saúde Atlântica Sports Center – F.C. Porto Stadium, Minho
University and Porto University Research Center, Portugal;
4Orthopedic Department Centro Hospitalar Póvoa de Varzim –
Vila do Conde, Portugal; 5Life and Health Science Research
Institute (ICVS), School of Health Sciences, University of Minho,
Campus de Gualtar, Braga, Portugal; 6Molecular Oncology
Research Center, Barretos Cancer Hospital, Barretos, São Paulo,
Brazil
Meniscus lesions are among the most common orthopaedic injuries
which can ultimately lead to degeneration of the knee articular carti-
lage. The human meniscus has a limited healing potential, partly due
to a poor vasculature, and thus meniscus regeneration using tissue
engineering strategies has recently been investigated as a promising
alternative to total/partial meniscectomy [1]. Advanced scaffolds for
tissue engineering of meniscus should be able to mimic and preserve
the asymmetric vascular network of this complex tissue, i.e. enable con-
trolling the segmental vascularization during the regeneration process.
Novel scaffolds were produced combining a silk polymeric matrix (12
wt%) [2] and the methacrylated gellan gum hydrogel (iGG-MA),
which has been shown to be able to prevent the ingrowth of endothe-
lial cells and blood vessels into the hydrogels [3,4]. The angiogenic/
anti-angiogenic potential of acellular and cell-laden silk-12 scaffolds
combined with iGG-MA hydrogel was investigated in vivo, using the
chick embryo chorioallantoic membrane (CAM) assay. For producing
the cell-laden scaffolds, human meniscus cells (HMC¢s) were isolated
from morphologically intact human menisci using an enzymatic-based
digestion and expanded using standard culture conditions. The HMC’s-
laden hydrogel/silk scaffolds were produced by encapsulating the
HMC’s into a 2 wt% GG-MA hydrogel, followed by impregnation onto
the 12 wt% silk scaffold and ionic-crosslinking in a saline solution. A
CAM assay was used to investigate the control of segmental vasculari-
zation of the acellular and HMC¢s-laden hydrogel/silk scaffolds by the
effect of GG-MA hydrogel, until day 14 of embryonic development.
The in vivo study allowed investigating the number of macroscopic
blood vessels converging to the implants. The evaluation of possible
inflammation and endothelial cells ingrowths was performed by histo-
logical (haematoxylin and eosin - H&E - staining) and immunohisto-
chemical methods (SNA-lectin staining). When the silk-12 scaffold was
combined with the hydrogel, an inhibitory effect was observed as dem-
onstrated by the low number of convergent blood vessels. Results have
shown that iGG-MA hydrogel prevented the endothelial cells adhesion
and blood vessels infiltration into the HMC’s hydrogel/silk scaffolds,
after 4 days of implantation. This study showed that the hydrogel/silk
scaffolds enabled controlling the segmental vascularization, thus it can
possibly mimic the native vasculature architecture during meniscus
regeneration.
References:
1. H. Pereira, et al., Arthroscopy, 2011, 27(12):1706–1719.
2. L.-P. Yan, et al., Acta Biomater, 2012, 8(1):289–301.
3. J. Silva-Correia, et al., J Tissue Eng Regen Med, 2011, 5(6):e97-
e107.
4. J. Silva-Correia, et al., Tissue Eng Part A, 2012, 18(11–12):1203–
1212.
 2012 The Authors J Tissue Eng Regen Med 2012; 6 (Suppl. 2): 8–39.
Journal of Tissue Engineering and Regenerative Medicine  2012 John Wiley & Sons, Ltd. DOI: 10.1002/term.1608
18 Abstracts List


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Mechanical performance and biocompatibility study of methacrylated Gellan gum hydrogels with potential for nucleus pulposus regeneration

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