Adipose tissue regeneration in a 3D-printed poly(lactic acid) frame-supported space in the inguinal region of rats

Abstract

BACKGROUND:

Adipose tissue engineering has been studied as an alternative to current options for breast reconstruction, such as lipofilling, flap reconstruction, and silicone implants. Previously, we demonstrated that a poly(L-lactic acid) mesh containing a collagen sponge, containing neither cells nor growth factors, could be filled with the regenerated adipose tissues when implanted in rodent models. However, the main factor contributing to adipogenesis remained unclear.

OBJECTIVE:

We aimed to clarify whether adipogenesis can be achieved by the space provided by the mesh or by the bioactivity of collagen.

METHODS:

A three-dimensional (3D) poly(lactic acid) (PLA) frame, which was stiff enough to maintain its shape, was fabricated by 3D printing. The frame with (PLA+ColI) or without (PLA only) a type I collagen hydrogel was implanted in the inguinal region of rats for up to 12 months. Adipose tissue regeneration in the PLA only and PLA+ColI groups was evaluated histologically.

RESULTS:

The 3D PLA frame maintained its structure for 12 months in vivo and oil red O (ORO)-positive adipose tissues were regenerated in the frame. No significant difference in the ORO-positive area was detected between the PLA only and PLA+ColI groups.

CONCLUSION:

The space supported by the frame was a key factor in adipogenesis in vivo.

Keywords

Adipogenesis 3D printing PLA collagen hydrogel internal space

1. Introduction

Breast cancer is the most commonly diagnosed cancer among females worldwide and 2.1 million women were newly diagnosed in 2018 [1]. Most patients who receive mastectomy choose to undergo breast reconstruction to prevent psychological stress. The current options for breast reconstruction are autologous fat injection (i.e., lipofilling), autologous flap reconstruction, and silicone implants. However, these methods have several limitations. In autologous fat injection, graft volume decreases to approximately 30–80% [2,3] likely because of mechanical stress [3]. Autologous flap reconstruction is accompanied by donor site morbidity [4]. In silicone implants, there is a perceived risk of anaplastic large-cell lymphoma and encapsulation [5,6].

Adipose tissue engineering has been proposed as an alternative to the current options of breast reconstruction. Most studies on adipose tissue engineering utilize cells (e.g., adipose-derived stromal cells and mature adipocytes) and/or growth factors (e.g., basic fibroblast growth factor and vascular endothelial cell growth factor) in combination with scaffolds [3,7]. In contrast, we reported that a poly(L-lactic acid) (PLLA) mesh containing a collagen sponge, with neither cells nor growth factors, can be filled with regenerated adipose tissues when implanted in rats and rabbits [8,9]. We have studied the biodegradability and various properties of PLLA [10–12] and PLLA derivatives as regenerative scaffolds [13–15] or drug carriers [16,17]. Based on the degradability with mild inflammatory responses, this implant composed of PLLA and collagen could be substituted by the regenerated adipose tissues [8,9]. The collagen sponge increased the dimensional stability of the PLLA mesh-based implant and maintained its space in vivo. In contrast, the PLLA mesh without the sponge was compressed, resulting in less adipose tissue regeneration [8]. These findings motivated us to clarify whether the space itself provided by the implant or the tissue affinity of collagen is the primary factor in adipose tissue regeneration.

Recent progress in three-dimensional (3D) printing, a form of additive manufacturing technology, has allowed us to prepare implants with desired size [18]. In this study, we used a 3D-printed poly(lactic acid) (PLA) frame [19,20] for adipose tissue regeneration. This frame was expected to maintain a space in vivo without the use of matrices inside because the frame was much stiffer than the PLLA mesh with the collagen sponge [8], showing unchanged shape at a compression force of 1.1 N. In addition, the combination of 3D printing and computer-aided design enables us to adjust the size and shape of the 3D-printed frame according to the tissue defect after surgery. We used a collagen type I (ColI) hydrogel as a matrix to fill the frame, since ColI scaffolds have been shown to support adipose-derived stem cell attachment and adipogenesis [21] and promote cell infiltration in vivo [22]. A 3D PLA frame without or with ColI hydrogel (PLA only or PLA+ColI, respectively) was prepared and implanted inguinally in rats. Adipose tissue regeneration inside the implants was evaluated histologically.

2. Materials and methods

2.1. Ethics statement

All animal experiments in this study were performed in accordance with the animal experiment guidelines of the National Cerebral and Cardiovascular Center (NCVC) Research Institute and were approved by the Committee of Laboratory Animals of the NCVC Research Institute (permit no. 18033). All efforts were made to minimize pain and suffering experienced by the animals.

2.2. Preparation of implants

The implants were prepared as described previously [19,20,23] with minor modifications. A 3D frame (6 × 6 × 3 mm³) was created using the 123D Design software (Autodesk, CA, USA). The 3D PLA frames were fabricated using a 3D printer (Replicator^{^TM} 2X; MakerBot Industries, NY, USA), sterilized by immersion in 70% (w/w) ethanol for 20 min, washed three times with autoclaved ultrapure water, and air-dried. ColI hydrogels (2.4 mg/mL; Cellmatrix^{^TM} type I-P; Nitta Gelatin, Japan) were formed in the sterilized 3D PLA frame in accordance with the manufacturer’s protocol (Fig. 1).

Fig. 1.

(A) Digital 3D frame designed with the Autodesk 123D Design software. (B) Photograph of 3D PLA frames with (right) or without (left) ColI hydrogel. Scale bar = 1 mm.

2.3. Compression test on 3D PLA frame

A compression test on the 3D PLA frame was performed as previously described [19,24] with minor modifications. A sample was placed on the measuring plate of a compression tester (MCR301; Anton Paar, Austria) equipped with a 12 mm diameter load plate and was compressed at a head speed of 1% strain per second at 25 °C. The strain at a compression of 1.1 N was determined as the compression rate. Three different 3D PLA frames were tested (n = 3).

2.4. Inguinal implantation in rats

Implantation was performed as described previously [8,19] with minor modifications. Sprague Dawley rats (male; 7 weeks old; 295–335 g; Japan SLC, Japan) were anesthetized with a mixture of isoflurane (2%) and air (98%). A subcutaneous pocket was made on each inguinal region of the animals using surgical scissors, and an implant was placed into the pocket. Then, the pocket was sutured with four stitches using 3-0 silk (Ethicon, NJ, USA) and the animals were allowed to recover. The rats were kept in a temperature-controlled room at 22 °C in a 12 h light-dark cycle. Water and a pellet diet were provided ad libitum. Four to six implants were evaluated at each time point (n = 4 to 6).

2.5. Histological evaluation of implants

At 3, 6, and 12 months post-implantation, the rats were anesthetized and the implants were extracted. The implants were split into halves at the middle of the frame using a scalpel and fixed in 10% neutral-buffered formalin. The implants were subjected to hematoxylin/eosin and oil red O (ORO) staining at the Applied Medical Research Laboratory (Japan). Images of the stained implants were acquired using a digital microscope (Eclipse TE300; Nikon, Japan). In the ORO-stained images, the percentage of ORO-positive tissue area in the implants was quantified using ImageJ (National Institute of Health, MD, USA).

2.6. Statistical analysis

Differences between the ORO-positive areas at 3, 6, and 12 months post-implantation were evaluated by two-way analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant.

3. Results

Figure 1 shows the digital design of the 3D frame and implant constructs with or without ColI. The compression rate of the 3D PLA frame at 1.1 N was 0.45 ± 0.08% (mean ± standard deviation), suggesting that the frame was not deformed by the compression force. This value was much smaller than that of the PLLA mesh containing the collagen sponge (approximately 5%) [8].

Figure 2 represents typical photographs of implants at their enucleation 3, 6, and 12 months post-inguinal implantation in rats. All implants were covered with adipose tissues composed of microvascular networks. No obvious difference was observed between the PLA only and PLA+ColI groups, and the 3D PLA frame maintained its shape even after the 12 month implantation.

Fig. 2.

Photographs of implants at their enucleation at 3, 6, and 12 months post-inguinal implantation in rats.

Histological images of the implants are shown in Fig. 3. Evidently, regenerated tissues were formed in the PLA only and PLA+ColI implants at 3 months post-implantation. At 3 months, over half of the area of the 3D PLA frame was filled with ORO-positive adipose tissue. Cell aggregates and/or collagen fibers were observed in the other areas, but the proportion of non-adipose areas decreased with implantation period. At 12 months, the implants were almost completely filled with adipose tissue.

Fig. 3.

Photographs of implants subjected to (A) hematoxylin/eosin and (B) ORO staining. PLA frames with (PLA+ColI) or without (PLA only) the ColI hydrogel were implanted inguinally in rats for 3, 6, and 12 months. Scale bar = 1 mm.

Fig. 4.

Time-dependent change in the percentage of ORO-positive area in the implants. Data are shown as the mean ± standard deviation, n = 4, except for data of the PLA only group at 3 months (n = 5) and PLA+ColI group at 6 months (n = 6). No significant difference was detected by two-way ANOVA.

Figure 4 shows the time-dependent change in the percentage of ORO-positive tissue area (i.e., adipose tissues) in the 3D PLA frame. There was no significant difference between the PLA only and PLA+ColI groups. The area of the PLA only group was already saturated at 3 months post-implantation and tended to be larger than that of the PLA+ColI group at 3 and 6 months. In contrast, the area of the PLA+ColI group increased with implantation time and was as large as that of the PLA only group at 12 months.

4. Discussion

The 3D PLA implant frame used in this study was much stiffer than the PLLA mesh with the collagen sponge [8]. In addition, the shape of the frame remained unchanged even after the 12-month implantation period. Therefore, the 3D PLA frame was a good candidate to evaluate whether adipose tissue regeneration can be achieved in the space provided by the frame without any matrix in vivo.

Regenerated adipose tissues were formed in both PLA only and PLA+ColI implants and there was no significant difference in ORO-positive area between them. Although it was difficult to distinguish between the remaining ColI hydrogel network and the collagen fibers in the regenerated tissues, the hydrogel in the frame might degrade within three months. This phenomenon is supported by studies reporting that the collagen hydrogels degraded completely in two weeks when subcutaneously implanted in rats [22] and that collagen sponges degraded in three months in the inguinal regions of rats [8] and rabbits [9]. The PLA+ColI group showed a time-dependent increase in ORO-positive area. In contrast, the ORO-positive area of the PLA only group was saturated at 3 months was a slightly greater than that of the PLA+ColI group at 3 and 6 months. These results indicate that matrices filling the frame are not indispensable for adipose tissue regeneration if the frame withstands compression in vivo and maintains its internal space.

The combination of a fat flap and an acrylic chamber for the regeneration of large areas of adipose tissue has been studied clinically by Morrison et al. [25], where adipose-like tissues with a volume of 210 mL were formed in one of five patients. The chamber was likely used to maintain an internal space to support angiogenic sprouting, tissue growth, and expansion [26]. The 3D PLA frame might produce similar effects for adipose tissue regeneration. In contrast to the non-biodegradable chamber, which needed to be removed at 6 or 12 months post-implantation [25], the materials of our implant (PLA and ColI) were biodegradable and thus, the implant may not need to be removed after implantation. Since non-biodegradable silicone implants for breast augmentation have been recalled by the US Food and Drug Administration because of a risk of implant-associated anaplastic large-cell lymphoma [27], we believe that our biodegradable implants can be an alternative to the silicone implants. Investigation of adipose tissue regeneration using a biodegradable implant in large animals (i.e., miniature pigs) are in progress.

5. Conclusions

In this study, we showed that a frame that maintains an internal space in vivo supports adipose tissue regeneration even if no matrix is contained within. Regenerated adipose tissues that were comparable or superior to those formed in the PLA+ColI group were formed in the 3D PLA frame without any matrix.

Footnotes

Acknowledgements

The authors thank Dr. Kyoko Shioya and the staff of the Laboratory of Animal Experiments and Medicine Management, NCVC, for assisting with the care of the animals. This work was financially supported by AMED (grant no. JP19hm0102068).

Conflict of interest

None to report.

References

Wild

C.P.

Weiderpass

and Stewart

B.W.

, World Cancer Report: Cancer Research for Cancer Prevention, International Agency for Research on Cancer, Lyon, 2020.

Gir

Brown

S.A.

Oni

Kashefi

Mojallal

and Rohrich

R.J.

, Fat grafting: Evidence-based review on autologous fat harvesting, processing reinjection, and storage, Plast Reconstr Surg 130 (2012), 249–258.

Visscher

L.E.

Cheng

Chhaya

Hintz

M.L.

Schantz

J.T.

Tran

, Breast Augmentation and reconstruction from a regenerative medicine point of view: State of the art and future perspectives, Tissue Eng Part B 23 (2017), 281–293.

Man

L.X.

Selber

J.C.

and Serletti

J.M.

, Abdominal wall following free TRAM or DIEP flap reconstruction: A meta-analysis and critical review, Plast Reconstr Surg 124 (2009), 752–764.

Brody

G.S.

Deapen

Taylor

Pinter-Brown

House-Lightner

S.R.

Anderson

J.S.

, Anaplastic large cell lymphoma occurring in women with breast implants, Plast Reconstr Surg 135 (2015), 695–705.

Collett

D.J.

Rakhorst

Lennox

Magnusson

Cooter

and Deva

A.K.

, Current risk estimate of breast implant-associated anaplastic large cell lymphoma in textured breast implants, Plast Reconstr Surg 143 (2019), 30S–40S.

Tsuji

Inamoto

Yamashiro

Ueno

Kato

Kimura

, Adipogenesis induced by human adipose tissue derived stem cells, Tissue Eng Part A 15 (2009), 83–93.

Ogino

Morimoto

Sakamoto

Jinno

Yoshikawa

Enoshiri

, Development of a novel bioabsorbable implant that is substituted by adipose tissue in vivo, J Tissue Eng Regen Med 12 (2018), 633–641.

Ogino

Sakamoto

Lee

Yamanaka

Tsuge

Arata

, De novo adipogenesis using a bioabsorbable implant without additional cells or growth factors, J Tissue Eng Regen Med 14(7) (2020), 920–930.

10.

Fujiwara

Yamaoka

Kimura

and Wynne

K.J.

, Poly(lactide) swelling and melting behavior in supercritical carbon dioxide and post-venting porous material, Biomacromolecules 6 (2005), 2370–2373.

11.

Yamaoka

Takahashi

Ohta

Miyamoto

Murakami

and Kimura

, Synthesis and properties of multiblock copolymers consisting of poly(L-lactic acid) and poly(oxypropylene-co-oxyethylene) prepared by direct polycondensation, J Polym Sci Pol Chem 37 (1999), 1513–1521.

12.

Yamaoka

Takahashi

Fujisato

Lee

C.W.

Tsuji

Ohta

, Novel adhesion prevention membrane based on a bioresorbable copoly(ester-ether) comprised of poly-L-lactide and Pluronic (R): In vitro and in vivo evaluations, J Biomed Mater Res 54 (2001), 470–479.

13.

Yamaoka

Takebe

and Kimura

, Surface modification of poly(L-lactic acid) film with bioactive materials by a novel direct alkaline treatment process, Kobunshi Ronbunshu 55 (1998), 328–333.

14.

Kakinoki

Uchida

Ehashi

Murakami

and Yamaoka

, Surface modification of poly(L-lactic acid) nanofiber with oligo(D-lactic acid) bioactive-peptide conjugates for peripheral nerve regeneration, Polymers 3 (2011), 820–832.

15.

Kakinoki

Nakayama

Moritan

and Yamaoka

, Three-layer microfibrous peripheral nerve guide conduit composed of elastin-laminin mimetic artificial protein and poly(L-lactic acid), Front Chem 2 (2014), 52.

16.

Hsu

Y.I.

Masutani

Yamaoka

and Kimura

, Strengthening of hydrogels made from enantiomeric block copolymers of polylactide (PLA) and poly(ethylene glycol) (PEG) by the chain extending Diels-Alder reaction at the hydrophilic PEG terminals, Polymer 67 (2015), 157–166.

17.

Somekawa

Mahara

Masutani

Kimura

Urakawa

and Yamaoka

, Effect of thermoresponsive poly(L-lactic acid)-poly(ethylene glycol) gel injection on left ventricular remodeling in a rat myocardial infarction model, Tissue Eng Regen Med 14 (2017), 507–516.

18.

Pati

D.H.

Jang

Han

H.H.

Rhie

J.W.

and Cho

D.W.

, Biomimetic 3D tissue printing for soft tissue regeneration, Biomaterials 62 (2015), 164–175.

19.

Kambe

Murakoshi

Urakawa

Kimura

and Yamaoka

, Vascular induction and cell infiltration into peptide-modified bioactive silk fibroin hydrogels, J Mater Chem B 5 (2017), 7557–7571.

20.

Kambe

and Yamaoka

, Biodegradation of injectable silk fibroin hydrogel prevents negative left ventricular remodeling after myocardial infarction, Biomater Sci 7 (2019), 4153–4165.

21.

Mauney

J.R.

Nguyen

Gillen

Kirker-Head

Gimble

J.M.

and Kaplan

D.L.

, Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose derived mesenchymal stem cells with silk fibroin 3D scaffolds, Biomaterials 28 (2007), 5280–5290.

22.

Etienne

Schneider

Kluge

J.A.

Bellemin-Laponnaz

Polidori

Leisk

G.G.

, Soft tissue augmentation using silk gels: An in vitro and in vivo study, J Periodontol 80 (2009), 1852–1858.

23.

Kambe

Tokushige

Mahara

Iwasaki

and Yamaoka

, Cardiac differentiation of induced pluripotent stem cells on elastin-like protein based-hydrogels presenting a single-cell adhesion sequence, Polym J 51 (2019), 97–105.

24.

Kambe

Mizoguchi

Kuwahara

Nakaoki

Hirano

and Yamaoka

, Beta-sheet content significantly correlates with the biodegradation time of silk fibroin hydrogels showing a wide range of compressive modulus, Polym Degrad Stab 179 (2020), 109240.

25.

Morrison

W.A.

Marre

Grinsell

Batty

Trost

and O’Connor

A.J.

, Creation of a large adipose tissue construct in humans using a tissue-engineering chamber: A step forward in the clinical application of soft tissue engineering, EBioMedicine 6 (2016), 238–245.

26.

Findlay

M.W.

Dolderer

J.H.

Trost

Craft

R.O.

Cao

Cooper-White

, Tissue-engineered breast reconstruction: Bridging the cap toward large-volume tissue engineering in humans, Plast Reconst Surg 128 (2011), 1206–1215.

27.

US Food and Drug Administration. Allergan recalls Natrelle Biocell textured breast implants due to risk of BIA-ALCL cancer [updated 2019 December 9; cited 2020 June 1]. Available from: https://www.fda.gov/medical-devices/medical-device-recalls/allergan-recalls-natrelle-biocell-textured-breast-implants-due-risk-bia-alcl-cancer.