Scientists report on the construction of biodegradable star-shaped poly(l-lactic acid) (SS-PLLA) polymers capable of self-assembling into nanofibrous hollow microspheres that can be used as injectable cell carriers for cartilage repair. Using a number of animal models, the University of Michigan researchers demonstrated that the microspheres supported a greater degree and quality of cartilage regeneration than chondrocytes alone, nanofibrous, or solid core microspheres. Their developments are reported in Nature Materials in a paper titled, “Nanofibrous hollow microspheres self-assembled from star-shaped polymers as injectable cell carriers for knee repair.”
Biomaterials play a pivotal role in engineering tissue regeneration and repair, but to generate large pieces of tissue for transplantation in a hospital setting, a predesigned scaffold with the patient-specific anatomy is required, explain Drs. Xiaohua Liu, Xiaobing Jin, and Peter X. Ma. Injectable materials have advantages as less invasive approaches to repairing irregularly shaped defects and wounds in a clinic setting, but are not currently used for cartilage repair.
The University of Michigan team has developed a technology enabling their star-shaped SS-PLLA polymer building blocks to assemble into nano- and/or mesoscopic structures that can further be fine-tuned in terms of degradation rate and possible surface functionalities. They claim the nanofibers that make up the resulting hollow microspheres effectively mimic the structural features of collagen fibers, the main component of extracellular matrix. This feature allowed the team to hypothesize that the microspheres’ architecture would enhance cell-material interactions and promote cell migration, proliferation, and mass transport conditions to facilitate tissue regeneration and integration in a host.
For comparison, the team also generated nanofibrous PLLA microspheres and conventional solid PLLA microspheres. In vitro studies confirmed that chondrocytes readily latched on to both the nanofibrous hollow microspheres made from SS-PLLA and nanofibrous microspheres composed of PLLA with 100% attachment rate, whereas less than 60% of chondrocytes attached to solid-interior microspheres. Encouragingly, a significant number of cells was found to have migrated inside the nanofibrous hollow microspheres. Moreover, the chondrocytes seeded on both the nanofibrous hollow microspheres and the nanofibrous microspheres had significantly higher proliferation rates and levels of collagen-related gene expression.
To test the capacity of the nanofibrous hollow microspheres as an injectable scaffold to fill cartilage defects, nanofibrous hollow microspheres were mixed with chondrocytes and injected into a rat femoral condyle-shaped mold. After four weeks of incubation in vitro, a piece of new cartilage tissue with an identical shape to the rat femoral condyle was obtained. “These in vitro results demonstrated the capability of the nanofibrous hollow microspheres as an injectable scaffold to fill a complex cartilage defect or to be molded into a predesigned 3-D tissue shape,” the team notes.
The researchers then tested the ability of chondrocyte-loaded nanofibrous hollow microspheres to stimulate cartilage formation in vivo. Two experimental models were employed: subcutaneous injection into nude mice for ectopic cartilage formation, and rabbit osteochondral defect repair.
Firstly, chondrocytes were mixed with each of the three types of microsphere and injected into subcutaneous pockets of the nude mice. Mice injected with chondrocytes alone, without microspheres, were used as a control group. After eight weeks the average tissue mass formed from the nanofibrous hollow microspheres-chondrocytes group was 36.9% higher than that formed from the nanofibrous microspheres-chondrocytes group, 197.3% higher than that formed from the solid-interior microspheres-chondrocytes group, and 235% higher than that formed in the group injected with chrondrocytes alone.
Moreover, after eight weeks most of the nanofibrous hollow microspheres had degraded, and abundant cartilage-specific matrix had been deposited into the void spaces. In contrast, all of the nanofibrous microspheres and the solid-interior microspheres were still in the tissues at eight weeks.
The authors suggest that the hollow structure and higher degradation rate of the nanofibrous hollow microspheres probably provided additional space for matrix accumulation, facilitating cartilage tissue formation. Significantly, regenerated cartilage from nanofibrous hollow microspheres was closer to native cartilage in histological appearance than that formed by injection of either the nanofibrous or solid-interior microspheres.
The team then tested whether nanofibrous hollow microspheres could be used as a carrier for chondrocytes to regenerate hyaline cartilage in a rabbit osteochondral defect repair model. The comparators for this experiment were PEG-conjugated chondrocytes, and chondrocytes without a carrier.
Animals were given injections of the test suspensions into induced knee defects, and regenerated tissue constructs were collected and evaluated eight weeks later. For animals in the nanofibrous hollow microspheres-chondrocytes group, the regenerated tissue fully filled the defect, smoothly integrated with the host cartilage, and was glistening white in appearance. In the PEG-chondrocytes group, the regenerated tissue was white but there was clearly a gap between the regenerated tissue and the host tissue, the researchers note. In the chondrocytes alone group, the regenerated tissue was thin but had integrated more smoothly with the host tissue than the new tissue formed in the PEG-chondrocytes group.
Significantly, tissue regeneration in the nanofibrous hollow microspheres group was primarily hyaline cartilage that stained strongly for collagen type II, whereas tissue formed in the other two groups did not demonstrate the same level of collagen markers.
“The injectable nanofibrous hollow microspheres self-assembled from SS-PLLA are an excellent micro-carrier for chondrocytes to facilitate high-quality hyaline cartilage repair...and worthy of further investigation towards the aimed clinical application,” the authors conclude. The University of Michigan team aims in the future to expand their research to evaluating the technology as a carrier for other regenerative cells and the repair of different tissue.