Rationale for the design of 3D-printable bioresorbable tissue-engineering chambers to promote the growth of adipose tissue

Abstract

Tissue engineering chambers (TECs) bring great hope in regenerative medicine as they allow the growth of adipose tissue for soft tissue reconstruction. To date, a wide range of TEC prototypes are available with different conceptions and volumes. Here, we addressed the influence of TEC design on fat flap growth in vivo as well as the possibility of using bioresorbable polymers for optimum TEC conception. In rats, adipose tissue growth is quicker under perforated TEC printed in polylactic acid than non-perforated ones (growth difference 3 to 5 times greater within 90 days). Histological analysis reveals the presence of viable adipocytes under a moderate (less than 15% of the flap volume) fibrous capsule infiltrated with CD68+ inflammatory cells. CD31-positive vascular cells are more abundant at the peripheral zone than in the central part of the fat flap. Cells in the TEC exhibit a specific metabolic profile of functional adipocytes identified by 1H-NMR. Regardless of the percentage of TEC porosity, the presence of a flat base allowed the growth of a larger fat volume (p < 0.05) as evidenced by MRI images. In pigs, bioresorbable TEC in poly[1,4-dioxane-2,5-dione] (polyglycolic acid) PURASORB PGS allows fat flap growth up to 75 000 mm3 at day 90, (corresponding to more than a 140% volume increase) while at the same time the TEC is largely resorbed. No systemic inflammatory response was observed. Histologically, the expansion of adipose tissue resulted mainly from an increase in the number of adipocytes rather than cell hypertrophy. Adipose tissue is surrounded by perfused blood vessels and encased in a thin fibrous connective tissue containing patches of CD163+ inflammatory cells. Our large preclinical evaluation defined the appropriate design for 3D-printable bioresorbable TECs and thus opens perspectives for further clinical applications.

Conflict of interest statement

PMD, PG and PM are co-inventors of a patent application that covers the design of the TEC and are consultants for Lattice Medical.

Figures

Figure 1

Material characterization and construction of TEC model (a) DSC curves of non-printed monofilaments of PLA (black) and PURASORB PGS (red) at a heating rate of 10 °C/min; (b) TGA curves of PLA (black) and PURASORB PGS (red); (c) Representative photomicrograph of TEC sample 3D-printed with PLA material for in vivo experiments.

Figure 2

Influence of TEC porosity on fat flap growth (a) (left) The dome-shaped, non-perforated chamber design with polylactic acid (pictures from above and below) (middle) MRI Quantitative analysis of in vivo growth kinetics of adipose tissue within non-perforated TEC. Volume is determined from MRI images. Results are means ± SD of replicate measurements by 3 different operators in 2 rats (rat 2 and 3); (right) Magnetic resonance imaging scans and 3D reconstruction of adipose tissue within non-perforated TEC at day 27 and 273 after implantation. The size increased of the Fat Flap (red) can clearly be seen. (White arrows indicate the chamber); (b) Diagram showing the configuration of the TEC containing 4 holes; Magnetic resonance imaging scans (Multiplanar reconstruction) of adipose tissue within the 4-hole TEC at days 10, 45 and 208 after implantation. Adipose tissue (red) has heterogeneously increased size with the fastest growth located in front of the holes. (Blue lines indicate the chamber); (c) Experimental design: schematic depicting follow-up of rats implanted with multiperforated TEC (TEC 3 see Table 2) by MRI and histological evaluations at the time points indicated (MRI were performed at day 76 only for rats 6 and 7 (d) (left) The dome- shaped, perforated chamber design with polylactic acid (pictures from above and below) and its insertion in rat; (middle) MRI quantitative analysis of in vivo growth kinetics of adipose tissue within the perforated TEC. Results are expressed as means ± SD of replicate measurements by 3 different operators in 4 separated rats (rat 4, 5, 6 and 7) (left) Magnetic resonance imaging in sagittal views of adipose tissue within the perforated TEC at day 13, 48, 76, 91 after implantation. The increased size of the Fat Flap (red) can clearly be seen. (White arrows indicate the limit of the chamber); (e) Comparison of in vivo growth kinetics of adipose tissue within non-perforated (n = 2) and perforated TECs (n = 4). Results are expressed as means ± SD of percentages of the theoretical volume within the TEC.

Figure 3

Histomorphometric analyses of fat flap under the multi-perforated TEC (a) Representative image of HE staining of new adipose tissue growing within the perforated TEC surrounded by connective tissue (left). Higher magnification view of a representative area under the connective tissue is shown on the upper right. Lower right Adipocyte surface area in the peripheral and central zone of the flap. Data are presented as Whiskers bar graphs (median 5–95 percentile). * p < 0.05; (b) Representative images of anti-CD31-stained sections of the chamber tissue. Higher magnification views of a representative area under the connective tissue are shown on the right part. (Red asterisks show CD31+ stained blood vessels). Lower left. Number of CD31+ cells in the peripheral and central zones of the flap. Data are mean + /SD * p < 0.05; (c) Representative image of Masson’s Trichrome stained tissue. Blue areas indicate collagen fibers deposition. (right) Note that adipose tissue components with septa were observed. Thickness of the connective tissue around the fat flap was measured. Data are presented as Whiskers bar graphs (median 5–95 percentile); (d) Representative images of anti-CD68-stained cells in the fat flap. Higher magnification views of a representative area in the connective tissue are shown. (Red asterisks show CD68 + stained macrophages). Lower left. Number of CD68+ cells in the connective and adipose tissues under the TEC. Data are mean + /SD * p < 0.05;

Figure 4

1H-NMR analysis of the metabolite profile of newly-generated adipose tissue in the TEC (a, b) Typical high-resolution 1D 1H-NMR spectrum of an extract of rat adipose tissue generated in the TEC (b) or of white adipose tissue serving as control (a, c) 2D COSY spectrum recorded from the adipose tissue in the TEC.

Figure 5

Influence of a TEC base on fat flap growth (a) Diagram showing the description of TEC4 and TEC5. The two tissue-engineering chambers (TEC 4 and TEC 5) differ by their dome porosity but have an identical flat base; (b) Growth curves of fat flap in the presence of TEC4 (left panel) or TEC5 (right panel) with (red line) or without a base (black line). Alternatively, maximum growth rates are calculated. Volume is determined on MRI images by three independent examiners. Values are expressed as mean ± SD (n = 3). * p < 0.05; (c) Representative longitudinal Magnetic Resonance Imaging (MRI) data of TEC4 with a base at day 20 (left) and day 80 (right) after implantation; (d) Representative longitudinal MRI data (left) and 3D modeling of fat flap (right) under TEC4 without a base at day 60 after implantation. White arrows show host tissues that fill the chamber impeding growth of the fat flap.

Figure 6

In vivo growth of fat flap under bioresorbable TEC (a) Surgical procedure of implanting the TEC in pig. The PGS growth chamber comprised of a flat base and a perforated dome-shaped lip. (Note the hole on the dome for the insertion of the pedicle vessels); lifting the pedicled adipose flap suture of the flap onto the base of the TEC with threads; suture of the dome on the base with threads; transferring the TEC and flap in the subcutaneous pouch and fixing on the underlying muscular aponeurosis; immediate postoperative aspect after watertight closure on 3 planes; (b) Experimental design: schematic depicting the follow-up of pigs implanted with bioresorbable multiperforated TEC (TEC 6 see Table 2) by MRI and histological evaluations at the indicated timepoints (MRI were performed at day 15 only for pigs 5, 6, 7, 8 (c) MRI quantitative analysis of in vivo growth kinetics of adipose tissue within bioresorbable TEC. Results are expressed as means ± SD in 8 pigs. Depending on growth rates we grouped pigs into 3 groups (Pigs 6 & 8, pigs 3, 5 and 7, pigs 2, 4 & 9). * p < 0.05; (d) Drawing of the set-up showing the pedicled adipose flap (yellow) inserted in the TEC in order to obtain adipose growth (red arrows); Gross morphology of the fat flap after insertion in the TEC; cross sections of fat tissues at day 90 after implantation. Note capsule of fibrovascular tissue at the peripheral of the TEC; Growth of adipose tissue within the bioresorbable TEC at days 15, 45 and 90 after implantation in pigs (n = 8). Results are expressed as means ± SD of percentages of the theoretical volume within the TEC * p > 0.05; (e) Comparison of relative growth rates of fat flap within the bioresorbable TEC and without the TEC. Results are expressed as means ± SD; (f) Comparison of relative growth rates of fat flap within bioresorbable TEC and weight of pigs. * p < 0.05; (g) MRI images in axial sections (T1 3D sequences) of fat flap within the TEC at day 15 (left panel) and day 90 (right panel). The fat flap within the TEC (red circle) is in hypersignal T1 with the TEC (white arrow, TEC visible at day 13 and not at day 83) being resorbed around it. In pig 6, an empty TEC without flap (blue circle) was used as control. Note the empty TEC without flap being resorbed then disappeared at day 83 leaving place to fibrosis; (h) Photograph showing a 90-day fat flap specimen (black star) within the bioresorbable chamber. Note that resorption was complete for the dome though some parts of the base had remained (white arrow). The figure was produced, in part, by using Servier Medical Art https://smart.servier.com/.

Figure 7

In vivo consequences of implanting the bioresorbable TEC (a) Evaluation over time of total serum protein and albumin:globulin ratio in pigs (n = 8) implanted within the bioresorbable TEC. Data are expressed as means ± SD. * p < 0.05 in comparison with results at day 0. Representative electrophoretic profiles of serum proteins from pigs implanted within the bioresorbable TEC at day 0 and day 15 post-implantation. The red arrow shows an alpha2 globulins peak; (b–d) Morphological evaluation of tissues harvested from the peripheral parts of the flap within the bioresorbable TEC at 90 days post-implantation; (b) Representative image of HE stained tissue, higher magnification view of connective tissue is shown on the right. Scales as marked. (lower) Whiskers plot (median 5–95 percentile) showing thicknesses of the fibrous capsule surrounding the adipose tissue; (c) Representative image of Masson’s Trichrome stained tissue; (d) Representative image of anti-α SMA+ stained cells in the connective capsule. (e) Representative images of anti-CD163-stained cells in the fat flap. (left and upper) Higher magnification views of positive representative areas in the connective tissue are also shown.

Figure 8

Morphological characterization of the adipose tissue within the bioresorbable TEC at day 90 post-implantation. (a) Representative images of HE stained adipose tissue harvested from the central and peripheral parts of the flap within the bioresorbable TEC at 90 days post-implantation; (b) left Perilipin-stained section of the adipose tissue within the bioresorbable TEC. Scale bar as indicated. (right) Quantification showed that the adipose tissue within the bioresorbable TEC had a higher adipocyte content than normal fat used as control (*p < 0.05); (c) Representative photographs of healthy, well-vascularized, adipose tissue with CD31+ capillaries within the bioresorbable TEC. Note also the well-vascularized connective tissue above fat tissue. Blood vessel walls are stained dark brown (red stars), and lumens with some containing red blood cells are visible at higher magnification. Comparison of the number of CD31+ cells per mm2 of fat flap within the bioresorbable TEC and without the TEC. Results are expressed as means ± SD.