*Article* **In Vitro Biocompatibility and Degradation Analysis of Mass-Produced Collagen Fibers**

**Kiran M. Ali, Yihan Huang, Alaowei Y. Amanah, Nasif Mahmood, Taylor C. Suh and Jessica M. Gluck \***

Department of Textile Engineering, Chemistry and Science, Wilson College of Textiles, NC State University, Raleigh, NC 27695, USA; kmmumtaz@ncsu.edu (K.M.A.); yhuang38@ncsu.edu (Y.H.); aya8@duke.edu (A.Y.A.); nmahmoo3@ncsu.edu (N.M.); tacook3@ncsu.edu (T.C.S.)

**\*** Correspondence: jmgluck@ncsu.edu; Tel.: +1-919-515-6637

**Abstract:** Automation and mass-production are two of the many limitations in the tissue engineering industry. Textile fabrication methods such as electrospinning are used extensively in this field because of the resemblance of the extracellular matrix to the fiber structure. However, electrospinning has many limitations, including the ability to mass-produce, automate, and reproduce products. For this reason, this study evaluates the potential use of a traditional textile method such as spinning. Apart from mass production, these methods are also easy, efficient, and cost-effective. This study uses bovine-derived collagen fibers to create yarns using the traditional ring spinning method. The collagen yarns are proven to be biocompatible. Enzymatic biodegradability was also confirmed for its potential use in vivo. The results of this study prove the safety and efficacy of the material and the fabrication method. The material encourages higher cell proliferation and migration compared to tissue culture-treated plastic plates. The process is not only simple but is also streamlined and replicable, resulting in standardized products that can be reproduced.

**Keywords:** collagen; tissue engineering; biomaterials; biocompatibility; scaffolds

### **1. Introduction**

Disease, injury, and trauma can damage and degenerate tissues in the human body that can be treated via the repair, replacement, or regeneration of the tissue [1]. Often, the tissues are either taken from the patient's own body (autograft) or from a donor (allograft). An example of an autograft is a bone graft, which is considered the "golden standard" treatment for spinal surgery [2]. Bone, tendons, cartilage, skin, heart valves, and veins are some of the tissues that can be treated using these methods [3]. This type of treatment has been revolutionary and has saved numerous lives. However, the treatment has many drawbacks, including cost and a sometimes difficult recovery, whereas a transplant may result in an immune response or even organ rejection [4].

For this purpose, tissue engineering is beneficial, as tissues are regenerated instead of being replaced. It involves the expansion of cells from a patient's biopsy with the help of ex vivo cell culture that function together to create tissues and organs. Tissue regeneration is carried out with the help of biological support in the form of engineered scaffolds, which restore or improve cell function [1]. Scaffolds play an important role in the manipulation of cell function and guidance toward new tissue development [5].

Various biomaterials have been used for the fabrication of scaffolds for regenerative medicine. Among them, collagen is one of the most prominent proteins due to its excellent biocompatibility [6]. Its high flexibility, optimal mechanical strength, and ability to absorb fluids, makes it an ideal biopolymer. Collagen is also one of the most prominent proteins found in the extracellular matrix (ECM) of humans and is expressed in tissues throughout the human body such as tendons, ligaments, bones, the dermis, dentins, and blood vessels [7–9]. Additionally, bovine and porcine collagens have shown degradable properties

**Citation:** Ali, K.M.; Huang, Y.; Amanah, A.Y.; Mahmood, N.; Suh, T.C.; Gluck, J.M. In Vitro Biocompatibility and Degradation Analysis of Mass-Produced Collagen Fibers. *Polymers* **2022**, *14*, 2100. https://doi.org/10.3390/ polym14102100

Academic Editors: Antonia Ressler and Inga Urlic

Received: 16 April 2022 Accepted: 18 May 2022 Published: 21 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

and have been used for the fabrication of biodegradable medical devices/scaffolds [10,11], such as drug delivery systems [12], and in bone tissue repair [13]. In a clinical study by Charriere et al. [14], the immunological response to bovine collagen implants was evaluated. Out of 705 participants, only 2.3% of the patients showed an adverse reaction to the collagen implant. Another study by Keefe et al. [15] also showed that the adverse effects were only observed in 1–2% of the treated patients, which were absolved when the material was resorbed by the hosts. For this reason, collagen polymers, especially porcine and bovine collagen polymers, are widely used in regenerative medicine.

There are multiple fabrication methods for the creation of scaffolds, such as solvent casting/particle leaching, thermally induced phase separation (TIPS), and threedimensional (3D) printing [16]. Although these techniques have been very advantageous for the creation of scaffolds, they all have disadvantages such as a lack of reproducibility, low production efficiency, and reduced mechanical strength [16,17]. The most common problems with these methods are reproducibility, repeatability, and standardization.

Textile fabrication methods, such as ring spinning, knitting, weaving, and braiding, have been used for centuries. Most of these fabrication techniques, including for yarn and fabric production, have now been automated and standardized, resulting in efficiently reproducible products. Using these methods to produce scaffolds will provide us with the ability to mass-produce products that are identical and that can be created in a standardized manner [18].

Traditional textile manufacturing methods, including knitting, weaving, and electrospinning, have been used for scaffold fabrication. There is a resemblance between the structure of native ECM and textile fibers [18]. The yarn-like structure of ECM resembles the fibrous morphology of textile materials, which has been proven to be beneficial for cellular migration and proliferation [19,20]. The fibrous architecture of ECM helps to provide support to the cell by creating a mesh of collagen, elastin, and other proteins. It is because of the fibrous structure of ECM that the cells are able to form a communication/signaling network [20]. Additionally, textile methods also create 3D structures, which are desirable for cellular scaffolds in terms of cell proliferation and migration [21].

Different methods are available for spinning yarns, including the ring spinning, rotor spinning, wrap-spinning, and core-spinning methods [22] (Table 1). Among these, ring spinning is the most popular method for creating yarns using staple fibers (short fibers ranging from 10–500 mm in length). The spun yarns can be used to create knit, woven, or braided structures for tissue engineering applications.


**Table 1.** The advantages and disadvantages of different spinning methods.

Weaving provides strength to the yarn. A study by Gilmore et al. [26] used round and grooved cross-sectional fibers to create plain weave and satin weave scaffolds. The results suggested that the fabric structure has a significant influence on the resultant properties, especially on the permeability of the scaffold. Similarly, knitting provides elasticity and

strength to fibrous scaffolds. A study by Lieshout et al. [27] shows a comparison of knitted and electrospun scaffolds for aortic valves. Human myofibroblasts were cultured on both scaffolds over the course of 23 days. The results compared tissue formation, which was evaluated via confocal laser scanning microscopy. The study showed that the electrospun scaffold tore within 6 h, while the knit scaffold remained intact. Another study by Zhang et al. [28] explored the idea of using a circular knit machine to produce a small-caliber vascular graft. The scaffold had excellent mechanical properties (bursting strength, suture retention strength, and compliance) that were comparable to the coronary artery under normotensive pressure. *Polymers* **2022**, *14*, x FOR PEER REVIEW 2 of 13

> This study evaluates an effort to mass-produce collagen fiber scaffolds that can be fabricated via traditional textile processes (Figure 1). In this study, collagen yarns are produced using the ring-spinning method, which is a process of creating yarns by twisting fibers together and winding them on a bobbin. The method comprises four processes: carding, drawing, roving, and ring spinning (Figure 2). The purpose of carding and drawing is to clean and align the fibers. After drawing, the fibers are passed to a roving machine, which fuses the fibers together through twisting, creating a roving. A roving is a sliver of fibers that is larger in diameter than yarn. The roving is then passed to a ring spinning machine, which provides the fibers with more twist, reducing the diameter to convert them into a yarn [22]. As a result, yarns are stronger when they are fabricated via ring spinning than other methods, such as rotor spinning. Ring spinning also provides a high production rate and can be used for any type of fiber [23]. have been used for the fabrication of biodegradable medical devices/scaffolds [10,11], such as drug delivery systems [12], and in bone tissue repair [13]. In a clinical study by Charriere et al. [14], the immunological response to bovine collagen implants was evaluated. Out of 705 participants, only 2.3% of the patients showed an adverse reaction to the collagen implant. Another study by Keefe et al. [15] also showed that the adverse effects were only observed in 1–2% of the treated patients, which were absolved when the material was resorbed by the hosts. For this reason, collagen polymers, especially porcine and bovine collagen polymers, are widely used in regenerative medicine.

**Figure 1.** Experimental Design Schematic. Collagen fibers were provided by the Kaneka Corporation and spun to yarns using the traditional ring spinning method. The resulting yarns were analyzed for biodegradability and biocompatibility. The biodegradability of the material was analyzed via weight changes, tensile strength changes, and morphological analysis. The biocompatibility was checked with Alamar blue and live/dead analysis for cell viability, and SEM and phalloidin staining were used for cytoskeleton analysis. **Figure 1.** Experimental Design Schematic. Collagen fibers were provided by the Kaneka Corporation and spun to yarns using the traditional ring spinning method. The resulting yarns were analyzed for biodegradability and biocompatibility. The biodegradability of the material was analyzed via weight changes, tensile strength changes, and morphological analysis. The biocompatibility was checked with Alamar blue and live/dead analysis for cell viability, and SEM and phalloidin staining were used for cytoskeleton analysis.

There are multiple fabrication methods for the creation of scaffolds, such as solvent casting/particle leaching, thermally induced phase separation (TIPS), and three-dimen-

Textile fabrication methods, such as ring spinning, knitting, weaving, and braiding, have been used for centuries. Most of these fabrication techniques, including for yarn and fabric production, have now been automated and standardized, resulting in efficiently reproducible products. Using these methods to produce scaffolds will provide us with the ability to mass-produce products that are identical and that can be created in a standard-

Traditional textile manufacturing methods, including knitting, weaving, and electrospinning, have been used for scaffold fabrication. There is a resemblance between the structure of native ECM and textile fibers [18]. The yarn-like structure of ECM resembles

creation of scaffolds, they all have disadvantages such as a lack of reproducibility, low production efficiency, and reduced mechanical strength [16,17]. The most common prob-

lems with these methods are reproducibility, repeatability, and standardization.

ized manner [18].

**Figure 2.** Ring spinning process for collagen yarns. The collagen fibers were provided by the Kaneka Corporation. The fibers were first run through the card and drawer with multiple sets of rollers. The rollers combed the fibers to clean and aligned them before they were gathered to form a sliver in a can. The sliver then passed through a roving frame, where the sliver reduced in diameter to form a roving, which was wound on a bobbin. Finally, the roving bobbins were passed through a ring spinning machine, where their diameters were further reduced via twisting with the help of a traveler and a ring. **Figure 2.** Ring spinning process for collagen yarns. The collagen fibers were provided by the Kaneka Corporation. The fibers were first run through the card and drawer with multiple sets of rollers. The rollers combed the fibers to clean and aligned them before they were gathered to form a sliver in a can. The sliver then passed through a roving frame, where the sliver reduced in diameter to form a roving, which was wound on a bobbin. Finally, the roving bobbins were passed through a ring spinning machine, where their diameters were further reduced via twisting with the help of a traveler and a ring.

After the yarns were fabricated, they were subjected to biocompatibility and biodegradation analysis to evaluate the perspective use of this technique in the future to massproduce scaffolds. The biocompatibility analysis provides knowledge about the safety and efficacy of the fabricated when encountering cells. This is crucial for the application of this process in tissue engineering products, especially for dermal [29], cardiovascular [28], tendon [30], and ligament [18] applications. After the yarns were fabricated, they were subjected to biocompatibility and biodegradation analysis to evaluate the perspective use of this technique in the future to massproduce scaffolds. The biocompatibility analysis provides knowledge about the safety and efficacy of the fabricated when encountering cells. This is crucial for the application of this process in tissue engineering products, especially for dermal [29], cardiovascular [28], tendon [30], and ligament [18] applications.

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

Collagen fibers were provided by the Kaneka Corporation. The fibers were processed through a card chute feed system (Rieter Card C4) followed by two rounds of drawing (Rieter RSB851). The aligned fibers were then converted to a roving with a roving machine (Rieter Fly F4/1) and finally spun into yarn on a ring spinning machine (Rieter G5/2) (Figure 2). The yarns had a fineness of 20 Ne, which is the thickness or coarseness of a yarn, and were measured according to the English Cotton Count (Ne) method, which is defined as "the number of 840-yard length per pound" [23]. Tensile strength and ultrastructure characterization were carried out via scanning electron microscopy (SEM), and an in vitro biocompatibility analysis was performed on the resulting yarns (Figure 1). Collagen fibers were provided by the Kaneka Corporation. The fibers were processed through a card chute feed system (Rieter Card C4) followed by two rounds of drawing (Rieter RSB851). The aligned fibers were then converted to a roving with a roving machine (Rieter Fly F4/1) and finally spun into yarn on a ring spinning machine (Rieter G5/2) (Figure 2). The yarns had a fineness of 20 Ne, which is the thickness or coarseness of a yarn, and were measured according to the English Cotton Count (Ne) method, which is defined as "the number of 840-yard length per pound" [23]. Tensile strength and ultrastructure characterization were carried out via scanning electron microscopy (SEM), and an in vitro biocompatibility analysis was performed on the resulting yarns (Figure 1).

### *2.1. Degradation Study 2.1. Degradation Study*

A degradation study was conducted to check the ability of the material to degrade when implanted in the body or when used as an in vivo application. Collagenase is normally found in almost all mammalian tissues, including pig pancreas, beef pancreas, and human tissues. It helps in wound healing by moving the keratinocytes over the collagenrich dermis during re-epithelialization. A degradation study was conducted to check the ability of the material to degrade when implanted in the body or when used as an in vivo application. Collagenase is normally found in almost all mammalian tissues, including pig pancreas, beef pancreas, and human tissues. It helps in wound healing by moving the keratinocytes over the collagen-rich dermis during re-epithelialization.

A collagenase solution was prepared according to the protocol by Alberti et al. [31]. Collagenase isolated from *Clostridium histolyticum* cleaves the bonds between neutral

A collagenase solution was prepared according to the protocol by Alberti et al. [31]. Collagenase isolated from *Clostridium histolyticum* cleaves the bonds between neutral amino acids and glycine, which is found with a high frequency in collagen. This type of degradation is a simplistic method to predict the in vivo degradation profile of our ring-spun collagen yarns. First, a collagen yarn sample was dried, weighed, and placed in 12-well plates. In a separate glassware, 0.5 mL of 0.1 M TRIS-HCl and 0.005 M of CaCl<sup>2</sup> were mixed with 2 mg/mL of collagenase (Type I, powder, Gibco, >125 units/mg). A 2 mL amount of the prepared solution was added to each well such that each sample was immersed in the solution, and the samples were placed in an incubator at 37 ◦C for a total of 8 weeks. Their weight, tensile strength, and morphology were monitored biweekly. The ultrastructure and morphology of the yarns were assessed using SEM to evaluate the degradation pattern in the collagen yarns.

### 2.1.1. Weight Change

A total of 6 samples were prepared, all of which were 20 cm each in length. It was determined that the length would be longer to increase the accuracy. The samples were weighed and placed in a 12-well plate and immersed in collagenase solution for the entire length of the study. The samples were then placed in the incubator at 37 ◦C. The solution was maintained at a pH of 7–7.4 throughout the study. The samples were dried every 14 days by aspirating the solution and drying in the desiccator for 24 h. After drying, their weight was measured, and the samples were placed in the original well plates again and submerged in collagenase solution at 37 ◦C. Average mass loss was calculated with the following formula:

$$\text{Mass Loss} = \frac{\text{Original Mass} - \text{Final Mass}}{\text{Original Mass}}$$

### 2.1.2. Tensile Test

A total of 24 samples were prepared, each of which were 20 cm in length, and the samples were placed in 12-well plates. Collagenase solution was added to each well, and the samples were stored in the incubator at 37 ◦C. The pH of the samples was maintained at 7–7.4. Every 14 days, 6 samples were dried and were placed in the desiccator for 24 h. When they had dried completely, the samples were mounted on cardboard (as shown in Figure 3a). An MTS Criterion 43 Tensile tester (MTS Systems, Eden Prairie, MN USA) was used for the tensile test, and a 50 N load cell, a gauge length of 1 cm, and a crosshead speed of 10 mm/min were maintained for the tensile test.

### 2.1.3. Scanning Electron Microscopy

The samples were dried and mounted on stubs using double-sided carbon tape. They were sputter-coated with gold/palladium using a SC7620 Mini Sputter Coater (Quorum Technologies, East Sussex, UK) for 45 s, resulting in a 10 nm coating. Samples were imaged with a Phenom G1 desktop SEM (Phenom, ThermoFisher, Eindhoven, The Netherlands). A total of 9 samples were analyzed per time point, and 4 images were taken from each sample. The morphology of the yarns was observed at biweekly intervals. The images were analyzed for surface and bulk degradation.
