*Article* **Injectable Cell-Laden Polysaccharide Hydrogels: In Vivo Evaluation of Cartilage Regeneration**

**Yao Fu †,‡ , Sanne K. Both, Jacqueline R. M. Plass, Pieter J. Dijkstra , Bram Zoetebier and Marcel Karperien \***

Department of Developmental BioEngineering, Faculty of Science and Technology, Tech Med Centre, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands

**\*** Correspondence: marcel.karperien@utwente.nl; Tel.: +31-(0)53-4893323

† Present address: Department of Obstetrics, The Second Clinical Medical College, Jinan University (Shenzhen People's Hospital), Shenzhen 518001, China.

‡ Present address: Post-Doctoral Scientific Research Station of Clinical Medicine, Jinan University, Guangzhou 510632, China.

**Abstract:** Previously, 5% *w*/*v* hyaluronic acid-tyramine (HA-TA) and dextran-tyramine (Dex-TA) enzymatically cross-linked hybrid hydrogels were demonstrated to provide a mechanically stable environment, maintain cell viability, and promote cartilaginous-specific matrix deposition in vitro. In this study, 5% *w*/*v* hybrid hydrogels were combined with human mesenchymal stem cells (hMSCs), bovine chondrocytes (bCHs), or a combination of both in a 4:1 ratio and subcutaneously implanted in the backs of male and female nude rats to assess the performance of cell-laden hydrogels in tissue formation. Subcutaneous implantation of these biomaterials showed signs of integration of the gels within the host tissue. Histological analysis showed residual fibrotic capsules four weeks after implantation. However, enhanced tissue invasion and some giant cell infiltration were observed in the HA-TA/Dex-TA hydrogels laden with either hMSCs or bCHs but not with the co-culture. Moreover, hMSC-bCH co-cultures showed beneficial interaction with the hydrogels, for instance, in enhanced cell proliferation and matrix deposition. In addition, we provide evidence that host gender has an impact on the performance of bCHs encapsulated in HA-TA/Dex-TA hydrogels. This study revealed that hydrogels laden with different types of cells result in distinct host responses. It can be concluded that 5% *w*/*v* hydrogels with a higher concentration of Dex-TA (≥50%) laden with bCH-hMSC co-cultures are adequate for injectable applications and in situ cell delivery in cartilage regeneration approaches.

**Keywords:** injectable hydrogel; mesenchymal stem cells; chondrocytes; co-cultures; in vivo; subcutaneous implantation; cartilage regeneration

**1. Introduction**

Articular cartilage injuries may occur as a result of either traumatic mechanical destruction or progressive mechanical degeneration. The combination of lack of blood supply and low mitotic activity of chondrocytes leads to limited ability to repair and regenerate articular cartilage [1–4]. Currently, injectable in-situ-formed hydrogels have emerged as promising cartilage tissue engineering strategies due to the ability to form three-dimensional, highly hydrated scaffolds after injection in aqueous form [5–7].

Injectable materials enable localized and straightforward delivery of cells and biomolecules via minimally invasive procedures without the associated risks of surgical implantation. These materials allow for the ability to fill irregular-shaped defects, avoiding the difficulty of prefabricating patient-specific defect shapes [8,9]. Moreover, hydrogels with different physical properties can also be designed and implanted in non-self-healing critical-size defects, temporarily replacing the extracellular matrix and assisting the healing process [10]. Previously, our group developed an injectable hybrid hydrogel composed of tyramine-conjugated hyaluronic

**Citation:** Fu, Y.; Both, S.K.; Plass, J.R.M.; Dijkstra, P.J.; Zoetebier, B.; Karperien, M. Injectable Cell-Laden Polysaccharide Hydrogels: In Vivo Evaluation of Cartilage Regeneration. *Polymers* **2022**, *14*, 4292. https:// doi.org/10.3390/polym14204292

Academic Editor: Antonia Ressler

Received: 8 September 2022 Accepted: 9 October 2022 Published: 12 October 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/).

acid (HA-TA) and dextran (Dex-TA) [11]. This hydrogel is formed in situ using a biocompatible enzymatic cross-linking reaction and supports survival of chondrocytes (CHs) and mesenchymal stem cells (MSCs) and tissue formation in vitro [12].

Tissue exposure to biomaterials triggers a foreign body response, a non-specific immune response process, which may result in persistent chronic inflammation and fibrotic encapsulation of the material [13–15]. In this inflamed environment, macrophages, lymphocytes, and their granular products contribute to the infiltration of foreign body giant cells (multi-nucleated fused macrophages) into the implantation site and the development of a collagen-rich fibrotic connective tissue layer surrounding the implant [14,16–18]. Degree of host response depends on the extent of homeostasis that is disturbed in the host by the injury, the implantation of the foreign material, and the properties of the material itself. Previously, an implant was considered biocompatible if it was encapsulated by an avascular layer of collagen without affecting its intended performance [18]. However, the impermeable nature of fibrous capsules, in some cases, results in poor mass transport and electrolyte diffusion between cell-laden implants and tissue, which impairs function, safety, and biocompatibility [19–21]. This is particularly relevant when these constructs are used in a tissue regeneration strategy.

In a previous study, we demonstrated that 5% *w*/*v* tyramine-conjugated hyaluronic acid and dextran (HA-TA/Dex-TA) hybrid hydrogels laden with bCHs provide a mechanically stable environment, maintain cell viability, and promote a cartilaginous-specific matrix deposition in vitro [12]. In the current study, 5% *w*/*v* hybrid hydrogels were combined with human mesenchymal stem cells (hMSCs) and bovine chondrocytes (bCHs) and then subcutaneously implanted in the backs of male and female nude rats for a four-week period. The cell laden hydrogels have a storage modulus (G') of 1.9 kPa for pure Dex-TA, 3.2 kPa for 50/50 HA-TA/Dex-TA, and up to 9.6 kPa for pure HA-TA hydrogels [12]. The main objectives were to assess the response of cell-laden hydrogels with respect to tissue formation, characterize the reaction of neighboring tissues, and investigate the interaction between hybrid hydrogels and co-cultures or mono-cultures. Additionally, we investigated the effect of host gender differences on the outcomes of subcutaneous implantation of HA-TA/Dex-TA hydrogels laden with bCHs and hMSCs in the backs of male and female nude rats.

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

### *2.1. Materials*

Dextran (40 kDa, pharmaceutical grade) was purchased from Pharmacosmos, Holbæk, Denmark. Sodium hyaluronate (27 kDa, pharmaceutical grade) was purchased from Contipro Pharma, Dolní Dobrouˇc, Czech Republic. Tyramine (99%), DMF (anhydrous, 99.8%), LiCl (99.0%), p-nitrophenyl chloroformate (PNC, 96%), pyridine (anhydrous, 99.8%), DMSOd<sup>6</sup> (99.9%), NaCl (≥99.0%), D2O (99.9 atom % D), horseradish peroxidase (HRP, 325 U/mg solid), and hydrogen peroxide (30%) were purchased from Sigma-Aldrich, Schnelldorf, Germany. Tyramine·HCl salt (99%) was obtained from Acros Organics, Fair Lawn, NJ, USA. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 97%) was purchased from Fluorochem Ltd., Hadfield, UK. Ethanol (≥99.9%) and diethyl ether (≥99.7%) were purchased from Merck, Kenilworth, NJ, USA. Milli-Q water was used from the Milli-Q Advantage A10 system (Merck KGaA, Darmstadt, Germany) equipped with a 0.22 µm Millipak® 40 Express filter.

### *2.2. Cell Culture and Expansion*

bCHs were isolated from full-thickness cartilage knee biopsies from ~6-month-old calves according to the previously reported protocol [22]. bCHs were expanded in chondrocyte proliferation medium (Dulbecco's modified Eagle's medium (DMEM; Gibco, Billings, MT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 0.2 mM ascorbic acid 2-phosphate (ASAP; Sigma, St. Louis, MO, USA), 0.4 mM proline (Sigma, St. Louis, MO, USA), 1× non-essential amino acids (Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA)).

Human-bone-marrow–derived MSCs were isolated as previously reported [23] and cultured in MSC proliferation medium (α-MEM (Gibco) supplemented with 10% FBS (Gibco), 1% L-glutamine (Gibco), 0.2 mM ASAP (Sigma), 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), and 1 ng/mL bFGF)). The use of human material was approved by a local medical ethical committee. The medium was refreshed twice a week, and cells at Passage 3 were used for the experiments.
