**1. Introduction**

In the history of tissue engineering, degradable materials such as poly (lactic acid— PLA) and poly (glycolic acid—PGA) or their combination poly (lactide-co-glycolide— PLGA), being of smooth or rough surfaces, were capable of inducing tissue or organ regeneration [1]. Recently, degradable and non-degradable FDA-approved polymers [2] with bioactivated surfaces were shown boosting tissue engineering and regenerative medicine

**Citation:** Chor, A.; Takiya, C.M.; Dias, M.L.; Gonçalves, R.P.; Petithory, T.; Cypriano, J.; de Andrade, L.R.; Farina, M.; Anselme, K. In Vitro and In Vivo Cell-Interactions with Electrospun Poly (Lactic-Co-Glycolic Acid) (PLGA): Morphological and Immune Response Analysis. *Polymers* **2022**, *14*, 4460. https://doi.org/ 10.3390/polym14204460

Academic Editors: Antonia Ressler and Inga Urlic

Received: 29 June 2022 Accepted: 30 September 2022 Published: 21 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

applications [3,4]. To this end, medical device design, medical device implantation and immune responses play crucial roles, in combination, to modulate immune responses for tissue regeneration [5,6].

Concerning degradation products in the organism, PLA and PGA by-products have been considered to be nontoxic. After degradation, by-products of lactic acid and glycolic acid are similar to endogenous metabolites. Lactic and glycolic acid enter the tri-carboxylic acid cycle and are eliminated as carbon dioxide and water [7] through respiration, feces and urine [8].

Electrospinning is considered a smart and easy technique to produce 3D synthetic devices with a porous and fibrous architecture similar to biological extracellular matrix [9,10]. Particularly, this technique has been reported to be effective because it allows the mixture of different monomers to improve the degradation time for drug or cell delivery to a specific microenvironment [11]. Such devices are at the forefront in the regenerative medicine area improving cell behavior in a specific milieu. Specifically speaking, translational application of electrospun fibers for oral disease treatment has been used including a variety of oral clinical applications [12]. Regarding oral mucosa regeneration, Wang and colleagues [12] showed tissue engineering strategies using the electrospinning process. Such devices were applied in porcine, rabbits and dog models of oral mucositis induced by anticancer therapies. As there are few studies reporting the use of electrospun fibers for cell therapy to treat oral diseases [12], the present work intends to show how an electrospun PLGA construct, with the addition of fibroblast-like cells from hamster cheek paunch, can serve as a future dressing for chemotherapy-induced oral mucositis ulcerations in hamster model.

It is noteworthy the great potential of this technology to produce nanofibers or microfibers using an electric field. This field can be tunable to control the deposition of aligned or random micro or nanofibers onto a collector, in a 3D pattern, forming a biomimetic scaffold for tissue engineering applications [12]. In general, this system regards advantages, such as the possibility of obtaining a large surface area in relation to the volume, organized interfibrillar spaces, controlled malleability and the facility in generating materials with a variety of sizes, texture and shapes [13]. In addition, 3D electrospun materials keep properties that promote the flow of nutrients from the biological microenvironment, such as oxygenation and vessel penetration throughout the device, accurately as it happens in the human organism [12]. As a result, it runs accelerated cells proliferation [14].

Recently it was shown that, when covered with nanoparticles or bioactive factors, PLGA membranes can turn into a promising bifunctional scaffold [4]. In this way, those scaffolds play a crucial role in cell migration and differentiation, both critical for their future applications in the field of tissue engineering and regenerative medicine. Moreover, medical devices must be sterilized before animals or human applications. Although a variety of techniques can be used to sterilize medical devices, our group previously demonstrated, by physicochemical and structural analysis, that 3D electrospun PLGA membranes treated with gamma radiation showed preserved structures [15].

Over 50 years the term "biocompatibility" has been widely used to qualify the biological properties of a biomaterial. For instance, Liang et al. [16] reported that the selection and fabrications of biomaterials with different structures and forms, such as films, hydrogels, electrospun scaffolds, sponges and foams should be tested to enhance therapeutic efficiency. Accordingly, the present work poses special emphasis on in vitro tests to study cell–biomaterial interactions using MDCK cell-line and primary fibroblast-like cells isolated from hamster cheek paunch with manufactured electrospun PLGA membranes. Moreover, in the present study, tissue-biomaterial immune responses were analyzed after in vivo hamster implantation over time. To our knowledge, this is the first time that 3D electrospun PLGA membranes are studied in a hamster model.

Madin-Darby canine kidney (MDCK) cell-lines are epithelial cells used in experimental research models due to their potential to adhere and form either tubules or flat monolayer sheets in 3D and 2D structures, respectively. This multicellular architecture is formed in virtue of either intercellular interaction between epithelial cells or upon cell–matrix interactions in the appropriate scaffold [17]. On the other hand, primary fibroblast-like cells from hamster' cheek pouch were chosen by our group due to our future tissue engineering challenges, to test this construct in hamster models [18].

Biological responses of cell–material interactions are a function not only of biomaterials design or their intrinsic properties but also of the microenvironment. Specific and long-term reactions can differ as a function of the organ, tissue and or species [4,19–22]. In this milieu, the material's surface properties play an important role in modulating the immune system response, such as the foreign body reactions [23,24]. This immunological response has been analyzed in experimental models as being a natural response of the organism, which will remain as long as polymer fragments are present at tissue–material interfaces [25,26]. Such response recognized as foreign body response (FBR) is classified into two main types of multinucleated giant cells, the Langhans-type giant cells (LCs) and the multinucleated-type giant cells (MNGCs). The LC is present within granulomas of infectious and non-infectious etiology. Morphologically, LCs exhibit a circular or ovoid shape with nuclei arranged in the periphery of the cells, in a limited number (10–20 nuclei per cell), adopting a circular or horse-shoe pattern [27]. On the contrary, MNGCs result from the macrophage response to indigestible substances, exhibiting an irregularly shaped cytoplasm, which may contain hundreds of nuclei per cell [27].

Although PLGA is a widely used polymer in the area of regenerative medicine and tissue engineering, its processing in porous membrane thanks to electrospinning can modify the inflammatory reaction that it will induce and therefore its interaction with tissues [12]. The present study focused on the in vitro morphological analysis of cell–material interactions and on the in vivo immune response after PLGA electrospun membrane implantation. To process the aforementioned analysis over time, our group applied morphological, cytochemical and immunohistochemical approaches associated to light (conventional optical and laser scanning confocal) and electron (scanning and transmission) microscopy. These tools revealed cell proliferation over time onto the PLGA membranes in vitro, and tissue immune responses after membrane implantation, without fibrous tissue formation at the biomaterial interface.

### **2. Materials and Methods**
