Transformation of Breast Reconstruction via Additive Biomanufacturing

Abstract

Adipose tissue engineering offers a promising alternative to current breast reconstruction options. However, the conventional approach of using a scaffold in combination with adipose-derived precursor cells poses several problems in terms of scalability and hence clinical feasibility. Following the body-as-a-bioreactor approach, this study proposes a unique concept of delayed fat injection into an additive biomanufactured and custom-made scaffold. Three study groups were evaluated: Empty scaffold, Scaffold containing 4 cm(3) lipoaspirate and Empty scaffold +2-week prevascularisation period. In group 3, of prevascularisation, 4 cm(3) of lipoaspirate was injected into scaffolds after 2 weeks. Using a well-characterised additive biomanufacturing technology platform, patient-specific scaffolds made of medical-grade-polycaprolactone were designed and fabricated. Scaffolds were implanted in subglandular pockets in immunocompetent minipigs (n = 4) for 24-weeks. Angiogenesis and adipose tissue regeneration were observed in all constructs. Histological evaluation showed that the prevascularisation + lipoaspirate group had the highest relative area of adipose tissue (47.32% ± 4.12) which was significantly higher than both lipoaspirate-only (39.67% ± 2.04) and empty control group (8.31% ± 8.94) and similar to native breast tissue (44.97% ± 14.12). This large preclinical animal study provides proof-of-principle that the clinically applicable prevascularisation and delayed fat-injection techniques can be used for regeneration of large volumes of adipose tissue.

Figure 1. Overall concept of the prevascularisation and delayed fat injection concept.

Empty scaffold is first implanted at the breast region without the addition of any cells or growth factors. Over the next 2–3 weeks, connective tissue and vasculature invades within the scaffold volume forming a bed of capillaries within the pores. Fat is then injected into the pores of the scaffold. Owing to the presence of the pre-formed vascular bed would allow the fat to remain stable at the implantation sites.

Figure 2

(a) Scanning Electron Micrograph of the scaffold showing the struts, pores and pore-interconnections. (b–f) Implantation process of the scaffolds. (b) Liposuction procedure near the abdominal midline incision. (c,d) Process of injecting fat into the pores of the scaffold placed in the lipoaspirate only group. (c) shows an empty scaffold while (d) shows a completely filled scaffold. (e) shows the process of injecting fat into the prevascularisation + lipoaspirate group scaffolds. The scaffolds are placed empty into the implantation site and 2 weeks later, fat is injected into the scaffold pores while the scaffold remains implanted. (f) the final form of the scaffolds conforms highly to the natural breast shape (g) Physical and mechanical properties of the scaffolds.

Figure 3. Explantation images showing the integration of TECs with the host tissue.

Green arrow point out major blood vessels supplying blood to the TEC. (d,g) show empty scaffold-only group (e,h) show lipoaspirate-only group (f,i) show prevascularisation + lipoaspirate group. All scaffolds show good integration with the host tissues and large areas of fat (marked with +) and vascularisation (marked with ^¶) were observed qualitatively on all scaffolds.

Figure 4. (LEFT) Representative images showing H&E staining of tissue explanted from the empty scaffold group (superficial layers).

A majority of the tissue can be identified as being connective tissue and collagen with only very small patches of fat tissue. (RIGHT) Representative images showing H&E staining of tissue explanted from the empty scaffold group (deep layers). Adipose tissue is only seen at the edges of the construct and not in the central regions of the scaffold. Lymphatic structures (right panel, marked by red arrows) were also observed in all groups mainly localised near scaffold strands.

Figure 5. (LEFT) H&E stained sections of lipoaspirate-only group (superficial layers).

Overall, a higher percentage of fat tissue compared to overall tissue area, compared to empty scaffold group, was observed in this group. (RIGHT) H&E stained sections of lipoaspirate-only group (deep layers). Deeper layers of the scaffold showed lower relative adipose tissue areas and lower degrees of vascularisation.

Figure 6. (LEFT) H&E stained sections of prevascularisation + lipoaspirate group (superficial layers).

This group showed the highest accumulation of adipose tissue interspersed between connective tissue. Tissue morphology also showed similarities with native tissue. (RIGHT) H&E stained sections of prevascularisation + lipoaspirate group (deep layers). Adipose tissue area was the highest among all other groups. Adipose tissue regions seemed to be better connected to each other and formed interconnected structures.

Figure 7. Representative H&E-stained micrographs of regions around the scaffold strands showing non-specific minor granulomatose reactions.

(a) shows empty scaffold-only group, (b) shows lipoaspirate-only group (c) shows prevascularisation + lipoaspirate group. Yellow arrows point to macrophages.

Figure 8. Representative images of Masson’s Trichrome stained tissue sections.

Green indicates collagen fibres, red indicates muscle-fibres and dark brown shows cell nuclei. (a,d) show empty scaffold group (b,e) show prevascularisation + lipoaspirate group (c,f) shows lipoaspirate-only group. Besides the adipose tissue, a majority of the tissue filling the pores of the implant consisted of connective and smooth muscle tissue. The smooth muscle layers had the highest thickness in case of the prevascularisation + lipoaspirate group. (g) Graph showing the adipose tissue area relative to total tissue area (AA/TA) over 24 weeks. Negative control scaffold-only group had the lowest AA/TA (8.31% ± 8.94) which was significantly lower than lipoaspirate-only (39.67% ± 2.04), prevascularisation + lipoaspirate group (47.32% ± 4.12) and native breast tissue (44.97% ± 14.12) (p < 0.05, p < 0.01 and p < 0.01 respectively). No significant difference in AA/TA was observed between the other groups. (h) Graph showing blood vessel density in the tissue sections. Highest blood vessel density was observed in the prevascularisation + lipoaspirate group (38.01/mm² ± 2.02). However the density was not significantly higher than the scaffold-only (33.13/mm² ± 12.03), lipoaspirate-only (26.67/mm² ± 1.6) or control breast tissue (35.45/mm² ± 1.93). (i) Histogram showing the distribution of adipose cells by cell surface-area. All histograms were skewed to the right suggesting that majority of cell surface areas lay in 100–700 μm² range. The empty scaffold and lipoaspirate-only groups had a low number of adipose cells with surface areas larger than 800 μm². The prevascularisation + lipoaspirate group showed a more equalised distribution with a significantly large number of cells having a surface area larger than 1000 μm². (j) Graph showing tissue composition at week 24. TECs from empty scaffold group contained an estimated 4.99 cm³ ( ± 2.71) of adipose tissue, TECs from lipoaspirate-only group contained an estimated 23.85 cm³( ± 1.22) of adipose tissue, whereas TECs from prevascularisation + lipoaspirate group contained an estimated 28.391 cm³ ( ± 2.48) of adipose tissue. (k) Graph showing estimated fold increase in adipose volume compared to initial injected lipoaspirate volume (4 cm³). Prevascularisation + Lipoaspirate group had a higher fold increase in adipose volume (6.1 ± 0.62) compared to lipoaspirate-only group (4.95 ± 0.31); however, the difference was not statistically significant (p = 0.143).