*3.6. Histological Analysis*

The cell pellets with HA-AGMCs at the concentration of 170 µg mL−<sup>1</sup> was used for animal implant experiments to make in vivo chondrogenic analysis in an osteoarthritic mode. Osteoarthritis was surgically induced by anterior cruciate ligament transection (ACLT) and partial medial meniscectomy on one knee of male New Zealand rabbits. Half of the rabbit's femoral head are implanted with a magnet as a source of magnetic force as shown in Figure 8A. In this in-vivo experiment, the HA-AGMCs are pre-cultured with chondrocytes before implantation into rabbit OA knee. Subsequently, after 6 weeks of surgery, the rabbits received implants of pellets containing either only cells or HA-AGMCs with cells or nothing as control groups. *Polymers* **2019**, *11*, x FOR PEER REVIEW 13 of 16

**Figure 8.** (**A**) Half of the rabbit's femoral head implanted with a magnet as a source of magnetic force. H&E stain of the cartilage tissue treated with various groups of cells/magnet/HA-AGMCs for 4 weeks **Figure 8.** (**A**) Half of the rabbit's femoral head implanted with a magnet as a source of magnetic force. H&E stain of the cartilage tissue treated with various groups of cells/magnet/HA-AGMCs for 4 weeks in (**B**–**D**).

**4. Conclusions**  In summary, cartilage tissue-mimetic pellets have considerable advantages in over articular cartilage disorder obstacles. In this study, Am-HA-gelatin has been synthesized by modifying primary amino group on gelatin with hexanoic anhydride to obtain the amphiphilic property and then grafting hyaluronic acid on the amphiphilic gelatin which is capable to form simple core-shell hollow structure. The multifunctional HA-AGMC with specific targeting on CD44 receptors provided cartilage tissue-mimetic pellets with high structure stability and remained high cell viability even in the center of pellets after 14 days culture. Packing with superparamagnetic iron oxide After 4 weeks of implantation, the preliminary results revealed that control groups without HA-AGMCs displayed irregular layer of cartilage surface and degeneration of the cartilage tissue (as illustrated in Figure 8B). In addition, the abnormal growth and disorder of cartilage tissues were observed in pure cell without magnet and HA-AGMCs group shown in Figure 8C. More importantly, a newly formed tissue is evident in the group treated with a combination of chondrocytes with A-AGMCs and magnet in Figure 8D, which demonstrated that the cells treated with HA-AGMCs and magnetic force can improve the retention and biofunctionality of transplanted chondrocytes to form ordering arrangement in cartilage matrix, which is very important for cartilage repair. However, the comprehensive investigation about chondrogenic analysis and cartilage repair is in subsequent progress, which will be reported in the future.

#### nanoparticles (SPIOs) in the HA-AGMC microcapsule, the pellets with the magnetic HA-AGMCs **4. Conclusions**

primers used for PCR in chondrocytes.

version of the manuscript.

in (**B**–**D**).

demonstrated the combination of static magnetic field and magnet-derived shear stress can exhibit the highest cartilage tissue-specific gene expression. Our preliminary results have showed that the cells treated with HA-AGMCs and magnetic force exhibit the better growth and ordering of chondrocytes. In summary, cartilage tissue-mimetic pellets have considerable advantages in over articular cartilage disorder obstacles. In this study, Am-HA-gelatin has been synthesized by modifying primary amino group on gelatin with hexanoic anhydride to obtain the amphiphilic property and then grafting hyaluronic acid on the amphiphilic gelatin which is capable to form simple core-shell hollow

distribution profile of HA-AGMCs, Table S1: The protons on the primitive gelatin exhibited chemical shift signals from peak 1 to peak 12., Table S2: Characterization of the HA-AGMCs, Table S3: List of genes and the

**Author Contributions:** Conceptualization, S.-J.C. and S.-Y.C.; methodology, K.-T.H. and M.-Y.C; software, K.- T.H.; validation, T.-Y.L. and M.-Y.C.; formal analysis, K.-T.H.; investigation, M.-Y.C.; resources, C.-Y.C.; data curation, T.-Y.L. and C.-Y.C.; writing—original draft preparation, K.-T.H., S.-J.C. and S.-Y.C.; writing—review and editing, K.-T.H., T.-Y.L., C.-Y.C., S.-J.C. and S.-Y.C.; supervision, S.-J.C. and S.-Y.C.; project administration, S.-J.C. and S.-Y.C.; funding acquisition, S.-J.C. and S.-Y.C. All authors have read and agreed to the published

**Funding:** This research was funded by Ministry of Science and Technology, Taiwan, grant number MOST 107-

2221-E-214-012-MY3, MOST 108-2218-E-080-001, MOST 106-2221-E-009-065-MY3.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Supplementary Materials:** The following are available online at www.mdpi.com/xxx/s1, Figure S1: 1H NMR spectrum of molecules in D2O for gelatin., Figure S2: Quantification of gelatin-based molecules with TNBS. (A) structure. The multifunctional HA-AGMC with specific targeting on CD44 receptors provided cartilage tissue-mimetic pellets with high structure stability and remained high cell viability even in the center of pellets after 14 days culture. Packing with superparamagnetic iron oxide nanoparticles (SPIOs) in the HA-AGMC microcapsule, the pellets with the magnetic HA-AGMCs demonstrated the combination of static magnetic field and magnet-derived shear stress can exhibit the highest cartilage tissue-specific gene expression. Our preliminary results have showed that the cells treated with HA-AGMCs and magnetic force exhibit the better growth and ordering of chondrocytes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4360/12/4/785/s1, Figure S1: <sup>1</sup>H NMR spectrum of molecules in D2O for gelatin., Figure S2: Quantification of gelatin-based molecules with TNBS. (A) Absorption spectra of primary amino group of the gelatin reacted with TNBS at different concentrations. (B) Characterization of different degrees of substitution for the gelatin-based molecules., Figure S3: Particle size distribution profile of HA-AGMCs, Table S1: The protons on the primitive gelatin exhibited chemical shift signals from peak 1 to peak 12., Table S2: Characterization of the HA-AGMCs, Table S3: List of genes and the primers used for PCR in chondrocytes.

**Author Contributions:** Conceptualization, S.-J.C. and S.-Y.C.; methodology, K.-T.H. and M.-Y.C; software, K.-T.H.; validation, T.-Y.L. and M.-Y.C.; formal analysis, K.-T.H.; investigation, M.-Y.C.; resources, C.-Y.C.; data curation, T.-Y.L. and C.-Y.C.; writing—original draft preparation, K.-T.H., S.-J.C. and S.-Y.C.; writing—review and editing, K.-T.H., T.-Y.L., C.-Y.C., S.-J.C. and S.-Y.C.; supervision, S.-J.C. and S.-Y.C.; project administration, S.-J.C. and S.-Y.C.; funding acquisition, S.-J.C. and S.-Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Science and Technology, Taiwan, grant number MOST 107-2221-E-214-012-MY3, MOST 108-2218-E-080-001, MOST 106-2221-E-009-065-MY3.

**Conflicts of Interest:** The authors declare no conflict of interest.
