Development data associated with effects of stiffness softening of 3D-TIPS elastomer nanohybrid scaffolds on tissue ingrowth, vascularization and inflammation in vivo.

This DiB article contains data related to the research article entitled "Cellular responses to thermoresponsive stiffness memory elastomer nanohybrid scaffolds by 3D-TIPS" (Wu et al., 2018). Thermoresponsive poly (urea-urethane) nanohybrid elastomer (PUU-POSS) scaffolds were implanted in rats for up to 3 months. The porous structure and tensile mechanical properties of the scaffolds are listed and compared before and after in vitro and in vivo tests. The details of the histological analysis of the explants with different initial stiffness and porous structures at various time points are presented. The images and data presented support the conclusion about the coupled effects of stiffness softening and the hierarchical porous structure modulating tissue ingrowth, vascularization and macrophage polarization in the article (Wu et al., 2018).

Specifications table

Value of the data

Data presented in this article provide direct comparison of the stiffness softening and hierarchical structure of the 3D-TIPS scaffolds before and after in vitro and in vivo tests. The data magnify more insights about the changes of structures at multi-scales and mechanical properties of the scaffolds under biophysical and biological conditions.

The histological images of the scaffolds with different initial stiffness and porous structure by immunohistochemistry elucidate for the first time how the stiffness softening and digitally printed hierarchical porous structure regulate tissue ingrowth, vascularization and macrophage polarization towards an M2 phenotype at the early (week 4) and late (week 12) stages in vivo.

Data

Table 1 shows the stiffness softening effect of the scaffolds in vitro over day 0–28 and how they relax towards their intrinsic elasticity. The dimensions of the 3D printed preforms and the scaffolds as produced are shown in Table 2. Table 3, Table 4, Table 5, Table 6, Table 7 show the effects of softening during in vivo implantation at various time points, in terms of tensile mechanical properties and XRD characterization respectively. Fig. 1, Fig. 2, Fig. 3 depict low and high magnification of Hematoxylin and Eosin (H&E) and Masson׳s trichrome (M&T) staining showing collagen fibre orientation and tissue ingrowth within the explants. Table 8 quantifies the angiogenic response of the explants during implantation time with stiffness softening. The softening effects on macrophage polarization (M1 markers CD86, CD63 and M2 maker CD163) and T-cell response (markers CD3 and CD4) are quantified in Table 9, Table 10, Table 11, Table 12, Table 13, Table 14, Table 15; representative immunohistochemistry images are shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13.

Table 1

Stiffness softening of PUU-POSS scaffolds with 50% infill density, tested at wet condition before and after in vitro incubation at 37 over 28 days.

3D-TIPS scaffold, 50% infill		Tensile Modulus (at 50% strain) MPa	Tensile Modulus (at 100% strain) MPa	Ultimate tensile strength (breaking point), MPa	Strain at break, %	Toughness, J m−3× 104
50CC	Day 0	0.98 (±0.14)	0.82 (±0.21)	1.33 (±0.09)	179 (±8)	137 (±22)
	Day 28	0.45 (±0.08)	0.40 (±0.11)	0.77 (±0.15)	230 (±13)	115 (±20)
50CC+H	Day 0	0.53 (±0.02)	0.44 (±0.08)	0.76 (±0.05)	236 (±19)	113 (±27)
	Day 28	0.39 (±0.09)	0.32 (±0.08)	0.72 (±0.12)	240 (±18)	110 (±14)
50RTC+H	Day 0	0.44 (±0.06)	0.39 (±0.09)	0.67 (±0.03)	146 (±15)	146 (±12)
	Day 28	0.42 (±0.08)	0.38 (±0.10)	0.65 (±0.06)	149 (±19)	146 (±20)

Table 2

Dimensions of 3D-printed PVA preforms and PUU-POSS scaffolds made by 3D-TIPS.

Scaffold		x- Strut thickness (μm, n = 10)	y-Strut thickness (μm, n = 10)	z-Strut thickness (μm, n = 10)	Sample Size, (L×W×T, mm) (n = 6)	Apparent Volume (mm3)	Volume Swelling Ratio vs VPVA(%)
50% infill PVA preform (mould)		400	400	200	60.0 × 12.0 × 4.0	2880 ± 4
50CC	Wet, as produced, RT	197 ± 13	157 ± 9	118 ± 19	61.0 × 13.0 × 3.6	2855 ± 9	−0.9 ± 0.2
50CC+H	Wet, as produced, RT	176 ± 8	150 ± 8	121 ± 14	59.7 × 11.3 × 3.5	2361 ± 7	−18.0 ± 0.1
50RTC+H	Wet, as produced, RT	186 ± 10	140 ± 11	127 ± 10	58.9 × 12.7 × 3.9	2917 ± 13	1.2 ± 0.3

Table 3

Tensile modulus (at 50% strain) of the scaffold explants at weeks 4, 8 and 12.

Tensile modulus (MPa)	50CC	50CC+H	50RTC+H
Week 0	1.11 (±0.13)	0.77 (±0.09)	0.43 (±0.08)
Week 4	2.45 (±0.40)	2.13 (±1.38)	1.56 (±0.20)
Week 8	3.99 (±0.55)	3.73 (±0.78)	3.13 (±0.88)
Week 12	6.97 (±1.46)	6.08 (±1.35)	5.88 (±1.53)

Table 4

Strain at break of the scaffold explants at weeks 4, 8 and 12.

Strain at break (%)	50CC	50CC+H	50RTC+H
Week 0	179 (±18)	186 (±19)	146 (±15)
Week 4	340 (±24)	310 (±61)	291 (±70)
Week 8	444 (±73)	423 (±71)	406 (±122)
Week 12	521 (±70)	494 (±65)	454 (±80)

Table 5

Ultimate tensile strength (breaking point) of the scaffold explants at weeks 4, 8 and 12.

Ultimate tensile strength (MPa)	50CC	50CC+H	50RTC+H
Week 0	1.63 (±0.09)	0.99 (±0.05)	0.67 (±0.07)
Week 4	1.07 (±0.39)	1.01 (±0.45)	0.81 (±0.18)
Week 8	1.98 (±0.37)	1.86 (±0.53)	1.16 (±0.39)
Week 12	2.84 (±0.53)	2.60 (±0.75)	2.44 (±0.29)

Table 6

Toughness of the scaffold explants at weeks 4, 8 and 12.

Toughness (J m−3 104)	50CC	50CC+H	50RTC+H
Week 0	137 (±12)	146 (±12)	113 (±17)
Week 4	412 (±24)	370 (±66)	351 (±79)
Week 8	523 (±73)	463 (±81)	406 (±162)
Week 12	599 (±99)	524 (±77)	444 (±90)

Table 7

Analysis of WAXD spectra of the explants during implantation. Degree of crystallinity (Dc, %), d-spacing (d, A) of semicrystalline structure and broad halo peaks of amorphous structures.

Scaffolds		Week 0			Week 4			Week e8			Week 12
		2θ	d	Dc	2θ	d	Dc	2θ	d	Dc	2θ	d	Dc
50CC	Sharp peak 1	20.0	4.4	37.6
	Sharp peak 2	23.2	3.8
	Broad halo peak 1
	Broad halo peak 2										20.1
	Broad halo peak 3				30.0			30.5			31.2
	Broad halo peak 4				40.5			41.5			41.9
50CC+H	Sharp peak 1
	Sharp peak 2
	Broad halo peak 1
	Broad halo peak 2				19.2			19.2			20.0
	Broad halo peak 3	30.3			28.8			29.8			30.9
	Broad halo peak 4	41.3			42.1			42.2			42.2
50RTC+H	Sharp peak 1
	Sharp peak 2
	Broad halo peak 1
	Broad halo peak 2										19.3
	Broad halo peak 3	26.0			25.9			27.0			27.1
	Broad halo peak 4	42.3			42.0			42.7			41.6

Fig. 1

Hematoxylin & Eosin (H&E) stained histological structure of middle in-plane of 50CC scaffold explants at week 12 depicting tissue ingrowth within the scaffold network, ×2 magnifications.

Fig. 2

Subcutaneous implantation of 50CC+H scaffolds at week 12: (A) tissue integration of middle-in-plane of the 50CC+H scaffold by Hematoxylin and Eosin (H&E) staining, (B) collagen production by Masson׳s trichrome staining (M&T), (C) endothelial cell infiltration as identified by CD31 staining, used as a marker of angiogenesis; (D-F) enlarged views of middle-in-plane sections respectively. (G-I) Middle cross-sectional view and (J-L) enlarged middle cross-sectional view.

Fig. 3

Subcutaneous implantation of 50RTC+H scaffolds at week 12: (A) tissue integration of middle-in-plane of the 50RTC+H scaffold by Hematoxylin and Eosin (H&E) staining, (B) collagen production by Masson׳s trichrome staining, (C) endothelial cell infiltration as identified by CD31 staining, used as a marker of angiogenesis; (D-F) enlarged middle-in-plane views respectively. (G-I) Middle cross-sectional view and (J-L) enlarged middle cross-sectional view.

Table 8

Proportion of total tissue/scaffold volume occupied by blood capillaries at weeks 4, 8 and 12. Immunofluorescent staining of anti-CD31 marker for blood capillaries.

Capillary (%)	50CC	50CC+H	50RTC+H
Week 4	11 (±1)	6 (±2)	3 (±2)
Week 8	25 (±3)	12 (±4)	8 (±4)
Week 12	30 (±4)	20 (±5)	14 (±5)

Table 9

Host pan-macrophage/monocyte response (CD68+ marker) towards the implanted scaffolds in terms of numerical density (Nv), representing the number of cells across the scaffold per unit square (Nv/mm2) at week 4, 8 and 12 (n = 20 frames, 12 scaffolds in each group at each time point).

CD68+	50CC	50CC+H	50RTC+H
Week 4	353 (±54)	301 (±56)	210 (±46)
Week 8	322 (±48)	260 (±39)	164 (±48)
Week 12	228 (±39)	201 (±43)	115 (±52)

Table 10

Host macrophage response (CD86+ marker) towards the implanted scaffolds in terms of numerical density (Nv), representing the number of cells across the scaffold per unit square (Nv/mm2) at week 4, 8 and 12 (n = 20 frames, 12 scaffolds in each group at each time point).

CD86+	50CC	50CC+H	50RTC+H
Week 4	397 (±56)	289 (±47)	152 (±39)
Week 8	312 (±55)	224 (±51)	132 (±45)
Week 12	271 (±41)	186 (±55)	96 (±53)

Table 11

Host macrophage response (CD163+ marker) towards the implanted scaffolds in terms of numerical density (Nv), representing the number of cells across the scaffold per unit square (Nv/mm2) at week 4, 8 and 12 (n = 20 frames, 12 scaffolds in each group at each time point).

CD163+	50CC	50CC+H	50RTC+H
Week 4	360 (±64)	294 (±65)	78 (±36)
Week 8	531 (±88)	434 (±76)	103 (±67)
Week 12	679 (±94)	534 (±78)	167 (±46)

Table 12

Ratio of CD68+/ CD163+ of the various scaffold groups at weeks 4, 8 and 12.

CD68+/CD163+	50CC	50CC+H	50RTC+H
Week 4	0.98	1.02	2.69
Week 8	0.60	0.59	1.59
Week 12	0.33	0.38	0.68

Table 13

Ratio of CD86+/ CD163+ of the various scaffold groups at weeks 4, 8 and 12.

CD86+/CD163+	50CC	50CC+H	50RTC+H
Week 4	1.10	0.98	1.95
Week 8	0.59	0.52	1.28
Week 12	0.40	0.35	0.57

Table 14

Host T lymphocyte response (CD3+ marker) towards the implanted scaffolds in terms of numerical density (Nv), representing the number of cells across the scaffolds per unit square (Nv/mm2) at week 4, 8 and 12 (n = 20 frames, 12 scaffolds in each group at each time point).

CD3+	50CC	50CC+H	50RTC+H
Week 4	372 (±54)	301 (±56)	134 (±31)
Week 8	232 (±48)	204 (±39)	67 (±15)
Week 12	156 (±44)	109 (±43)	35 (±8)

Table 15

Host T lymphocyte response (CD4+ marker) towards the implanted scaffolds in terms of numerical density (Nv), representing the number of cells across the scaffolds per unit square (Nv/mm2) at week 4, 8 and 12 (n = 20 frames, 12 scaffolds in each group at each time point).

CD4+	50CC	50CC+H	50RTC+H
Week 4	301 (±61)	245 (±71)	152 (±27)
Week 8	252 (±42)	201 (±46)	102 (±28)
Week 12	122 (±32)	87 (±45)	32 (±16)

Fig. 4

Immunohistochemistry of the host macrophage response towards scaffolds in vivo at week 4. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD68 (M1 pan-macrophage/monocyte marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications.

Fig. 5

Immunohistochemistry of the host macrophage response towards scaffolds in vivo at week 12. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD68 (pan-macrophage/monocyte marker) staining at (A-C, G-I) ×4 and (D-F, J-I) ×20 magnifications. (M) Negative control (rat appendix); (N) positive control (rat liver). Scale bar: 100 μm.

Fig. 6

Immunohistochemistry of the host macrophage response towards scaffolds in vivo at week 4. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD86 (M1 macrophage marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications.

Fig. 7

Immunohistochemistry of the host macrophage response towards scaffolds in vivo at week 12. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD86 (M1 macrophage marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications. (M) Negative control (rat appendix); (N) positive control (rat liver). Scale bar: 100 μm.

Fig. 8

Immunohistochemistry of the host macrophage response towards scaffolds in vivo at week 4. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD163 (M2 macrophage marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications.

Fig. 9

Immunohistochemistry of the host macrophage response towards scaffolds in vivo at weeks 12. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD163 (M2 macrophage marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications. (M) Negative control (rat appendix); (N) positive control (rat liver). Scale bar: 100 μm.

Fig. 10

Immunohistochemistry of the host T lymphocyte response towards scaffolds in vivo at week 4. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD3 (T lymphocyte marker) staining at (A-C, G-L) ×4 and (D-F, J-L) ×40 magnifications.

Fig. 11

Immunohistochemistry of the host T lymphocyte response towards scaffolds in vivo at week 12. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD3 (T lymphocyte marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications. (M) Negative control (rat appendix); (N) positive control (rat spleen). Scale bar: 100 μm.

Fig. 12

Immunohistochemistry of the host T lymphocyte response towards scaffolds in vivo at week 4. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD4 (T lymphocyte marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications.

Fig. 13

Immunohistochemistry of the host T lymphocyte response towards scaffolds in vivo at week 12. Tissue integration of middle-in-plane (A-F) and cross-sectional view (G-L) of the scaffolds by CD4 (T lymphocyte marker) staining at (A-C, G-I) ×4 and (D-F, J-L) ×20 magnifications. (M) Negative control (rat appendix); (N) positive control (rat spleen). Scale bar: 100 μm.

Static tensile mechanical properties and hierarchical porous structure of the scaffolds

Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7.

Cellular infiltration and matrix deposition

Fig. 1, Fig. 2, Fig. 3.

Angiogenesis response

Table 8.

T-cell proliferative and host macrophage response

Table 9, Table 10, Table 11, Table 12, Table 13, Table 14, Table 15 and Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13.

Experimental design, materials and methods

Fabrication of thermoresponsive PUU-POSS scaffolds

A 3D-TIPS technique, based on reverse 3D printing and phase separation of the polymer solution, as described in [1], was used to manufacture PUU-POSS scaffolds (50% infill density) at different thermal conditions (50CC, 50CC+H and 50RTC+H).

Characterization of the scaffolds prior to implantation

An Instron 5655 was applied to test static tensile mechanical properties of the scaffolds, before and after incubation over 28 days at body temperature, as described in [1], as well the explants after implantation in rats for 4, 8 and 12 weeks. The dimensions of the printed preforms and the scaffold as produced were also measured and estimated.

Characterization of the scaffold explants

As detailed in [1], the scaffolds were subcutaneously implanted in adult male rats and harvested at different time points. The physico-mechanical properties (i.e. tensile properties and phase structure) were then analyzed with an Instron 5655 tester and an X-ray diffractometer. Sectioning and histological staining (i.e. H&E and M&T) were carried out, and collagen fiber formation and tissue ingrowth orientation was quantified as previously described [1]. Immunofluorescent staining against capillary marker CD31, macrophage markers CD86/CD68/CD163 and T-cell makers CD3/CD4 was carried out, and the number of positive stained cells was quantified as described in [1].

Subject area	Chemistry, biology
More specific subject area	Biomaterials
Type of data	Tables, Figures
How data were acquired	Static tensile mechanical testing (Instron5655), Mercury intrusion porosimeter (Quantachrome Poremaster 60GT), X-ray diffraction (XRD; Bruker D8 Advance), immunohistochemistry
Data format	Analyzed
Experimental factors	Scaffolds prior to implantation were subjected to uniaxial mechanical testing and mercury intrusion porosimeter. Scaffold explants at different time points were subjected to uniaxial mechanical testing and XRD characterized. In addition, explants were sectioned and stained for Hematoxylin and Eosin (H&E) and Masson׳s trichrome (M&T). Immunofluorescent staining was carried out to detect presence of capillary markers (i.e. CD31), macrophage markers (i.e. CD86, CD68, CD163) and T-cell makers (i.e. CD3, CD4).
Experimental features	Physico-mechanical characterization, histology and immunohistochemistry
Data source location	Centre for Biomaterials in Surgical Reconstruction and Regeneration, Division of Surgery & Interventional Science, University College London, Royal Free Hospital London NHS Foundation Trust, London, United Kingdom, NW3 2PF
Data accessibility	Within this article
Related research article	[1]L. Wu, A. Magaz, E. Maughan, N. Oliver, A. Darbyshire, M. Loizidou, M. Emberton, M. Birchall, W. Song, Cellular responses to thermoresponsive stiffness memory elastomer nanohybrid scaffolds by 3D-TIPS, Acta Biomater. (2018).doi:10.1016/j.actbio.2018.12.019.