According to the American Society of Gene & Cell Therapy, regenerative medicine may be defined as a branch of medicine that aims to repair damaged tissue. “Repair” may involve the replacement or regeneration of human cells, tissues, or organs. Either way, replacement or regeneration, the aim is to restore or establish a normally functioning whole—an elegant interweaving of tissue elements, not a flimsy, inelegant patch.

If regenerative medicine is to succeed and deliver much-desired cures, it will need to create living tapestries as closely woven and richly detailed as those found in natural, intact tissues. Such tapestries, it happens, are emerging from regenerative medicine’s workshops, which are employing diverse materials and realizing varied designs.

The materials include bioinks, microscale scaffolds (collagen matrices and vascular structures), extracellular vesicles, immunomodulatory and other signaling factors, and—of course—stem cells. All these materials are being incorporated into dermal fillers, cartilage, bone tissue, and even more ambitious works.

Soon, regenerative medicine will attempt cures for a vast array of disorders, including cancer, diabetes, cardiovascular disease, autoimmune diseases, and bone and joint inflammations. Already, the discipline’s looms are working toward 3D printed organs. Such masterpieces could end donor organ shortages while avoiding rejection issues.

Photocurable regenerative fillers

Type I collagen is the basic building block in connective tissues. To mass produce recombinant human collagen (rhCollagen), CollPlant Biotechnologies uses tobacco plants, genetically engineered with the five genes responsible for the protein’s synthesis.

The tobacco plant was selected as the host for collagen synthesis for three main reasons: the well-known tobacco genome facilitates manipulation; tobacco is not part of the food and feed chain; and the large biomass leaves can be harvested in 6–8 weeks and the plants reharvested through three growing cycles.

CollPlant’s rhCollagen is the basis of the company’s BioInk products, which are used for 3D bioprinting. To produce a BioInk, CollPlant begins by chemically modifying rhCollagen so that it undergoes crosslinking when exposed to light. Next, polymers are added to mimic the target tissue’s physical properties. Finally, biological constituents make each BioInk biologically tissue specific.

“Our rhCollagen is ‘virgin’ with many cell-binding domains,” says Yehiel Tal, CollPlant’s CEO, “and it shows zero immune response compared to products that are based on tissue-extracted collagen.”

“BioInks are injected through very fine nozzles,” he continues. “We reduce the shear force significantly during the deposition then return it to the original viscosity to maintain high cell viability. After deposition, our BioInks can be photocured quickly with accurate positioning while enabling high-production throughput.”

“We jointly develop products with leading companies and are also building a portfolio of medical aesthetic products in which the common denominator is a collagen-based formulation,” Tal adds.

The market-dominant dermal filler, hyaluronic acid (HA), is hydrophilic, has good skin-lifting capacity, and is easily injected with a reasonable longevity. But HA does not regenerate the tissue; collagen adds the biological properties necessary for tissue rejuvenation.

CollPlant is developing dermal fillers that are comprised of collagen and HA and that are showing good tissue integration and skin-lifting capacity in ongoing preclinical studies. These fillers may leverage the features developed for photocuring BioInks. That is, upon illumination with a flashlight, they may enable post-injection skin sculpting and in vivo crosslinking. Additional products in the pipeline include 3D bioprinted regenerative breast implants and an injectable implant to address breast-shaping applications.

Hololithographic bioprinting

High fidelity and resolution are two of the biggest challenges in bioprinting. To overcome these challenges, Prellis Biologics has developed the Holograph X, a bioprinter that leverages a multiphoton laser system to perform holographic stereolithography. The Holograph X, which Prellis Biologics is developing and commercializing in partnership with Cellink, can print structures with micron-level resolution. These structures include hollow, permeable channels that can mimic capillaries and ultrafine vessels.

“Tissues and organs need a biomimetic microvascular network to survive,” comments Erin Stephens, PhD, director of tissue engineering at Prellis Biologics. “Resolution is key to allowing sufficient nutrient and oxygen exchange.

“Our hologram-based process starts by printing a high-resolution scaffold, followed by seeding the scaffold with cells. The types of microfluidic devices you can print with hololithography are very different than those printed with extrusion printers. We can build different shapes, different topographies, and different flow networks.”

“At Prellis,” she adds, “we continually push the limits of resolution and speed. Cells and living tissue need the detail; they need the capillary beds. We can print 240,000 voxels (3D pixels) per second. Printing a cubic centimeter with 1-µm resolution and a 1% fill factor takes us 11 hours compared to about 3 years for other similar technologies currently on the market.”

Vascular Tissue Blanks
Prellis Biologics, a 3D tissue printing startup, uses laser and holographic printing techniques to build 3D hydrogel structures such as 3D capillaries, which the company calls Vascular Tissue Blanks. These structures, or “tissue chips,” have been used to culture cell types ranging from breast cancer cells to human primary liver cells. A typical Vascular Tissue Blank consists of a network of hollow tubes that is just 2 mm wide.

The required 3D scaffold architectures are organ dependent; that is, different organs have different flow requirements. Ideal organ-specific scaffolds have yet to be systematically investigated. To facilitate scaffold design, Prellis Biologics offers TissueWorkshop, a software platform that reduces the time required for scaffold design and iteration. TissueWorkshop accomplishes in seconds to minutes tasks that would keep conventional 3D modeling software busy for hours or days. The company also supplies Vascular Tissue Blanks if users need a starting point.

Stephens believes that the field will evolve with bursts of momentum that will drive new breakthroughs, and that interdisciplinary amalgamation will be critical to assemble all the information needed to get closer to true organ transplant. “Prellis has made huge strides in the field of regenerative medicine and that has already been incredibly impactful toward future healthcare,” he declares. “But there is still a lot to do.”

Prodding the body’s own stem cells

To replace or regenerate tissue, you could introduce new stem cells to a patient’s body. Alternatively, you could activate or stimulate the body’s own stem cells. The latter approach is being developed by Histogen. The company is focused on exposing a patient’s own stem cells to proteins and growth factors that have been produced by fibroblasts grown under simulated embryonic conditions, that is, in low oxygen and suspension cultures. The fibroblasts, which are induced to become multipotent stem cells, generate a liquid complex containing soluble biologicals with a diverse range of embryonic-like proteins. The biologicals can stimulate a patient’s own stem cells and allow regenerative applications to do animal-derived materials.

Histogen collects not only soluble proteins and growh factors, but also soluble and insoluble human extracellular matrix (hECM) materials. The hECM materials include components associated with stem cell niches in the body and scarless healing of fetal skin.

“Our in vitro studies demonstrated that the hECM supports the regulation and proliferation of human mesenchymal stem cells, as well as cell surface markers showing the maintenance of stemness, and induces the upregulation of aggrecan and Collagen II,” states Gail Naughton, PhD, Histogen’s founder and chief scientific officer. “These are important components in hyaline cartilage regeneration in damaged knees.”

Results from a goat model
Histogen produces human extracellular matrix (hECM) for applications in regenerative medicine. For example, the company’s joint cartilage product has been shown to regenerate mature cartilage and well-vascularized bone in preclinical models. Results from a goat model are shown here. In hECM-treated animals, defects continued to improve over 12 months with more mature hyaline cartilage and bone integrating with the adjacent normal tissue. The untreated defects at 12 months were characterized by large voids filled with fibrotic tissue and demonstrating insignificant evidence of subchondral bone and articular cartilage tissue regeneration.

In rat, rabbit, and goat models, hECM supported the regeneration of homogeneous hyaline cartilage and mature vascularized bone, whereas control knees showed only scar tissue formation. The tissue regeneration recapitulated the formation of cartilage, mineralization of the cartilage to form bone, and maturation of the cartilage to form a joint surface of a kind that is seen in embryogenesis.

The hyaline cartilage was fully attached to the adjacent cartilage, and a clear tide mark was seen. A 2020 U.S. clinical trial will study the hECM in regenerating hyaline cartilage in focal knee lesions.

The soluble hECM’s ability to reverse damage to degenerating spinal discs was also assessed. In a spinal disc study, a soluble extracellular matrix material reversed inflammation and protease activity and stimulated aggrecan secretion in a thrombin-induced ex vivo rabbit model. In vivo studies showed that within four weeks post treatment, disc height increased as compared to the control, and MRI analysis demonstrated regeneration of the disc tissue.

Histogen recently merged with Conatus Pharmaceuticals and is now listed on the NASDAQ exchange under HSTO.

Matrix-bound nanovesicles

In 2016, in the laboratory of Stephen F. Badylak, DVM, PhD, MD, small extracellular vesicles were seen to be physically embedded within the structural components of the extracellular matrix. This observation stimulated research that culminated in the characterization of entities now known as matrix-bound nanovesicles (MBVs).

Although MBVs are similar to fluid-phase extracellular vesicles, such as cellular-produced exosomes, the similarities between the two vesicle types end with size and shape. According to Raphael Crum, an MD-PhD candidate in Badylak’s laboratory, the characteristics that make these vesicles a unique population, and exciting for research, include their structure, function, and therapeutic potential.

Structurally, MBVs consist of a lipid membrane that transports cell signaling molecules, including microRNA (miRNA), (phospho)lipids, and proteins, that can influence processes such as macrophage activation, inflammation and fibrosis, tissue repair and remodeling, and wound healing. When MBVs are removed from their parent extracellular matrices, they recapitulate the same reconstructive remodeling and immunomodulatory properties seen with whole matrix biomaterials. MBV applications include more efficacious therapies for rheumatoid arthritis.

“Due to the broad immunomodulatory properties of MBVs and their cargoes, it is possible that MBVs might be used in the treatment of many other diseases and conditions stemming from a dysregulated immune response,” suggests Crum. The Badylak laboratory is currently investigating several other MBV applications in a variety of models of immune-mediated disease.

Crum believes as scientists develop a better understanding of the cellular and molecular biology involved in tissue regeneration in response to natural and synthetic biomaterials and cell-based therapies, the knowledge will facilitate the design of focused biomaterials that can operate in diverse patient and disease niches. Eventually, translational and personalized biomaterials will be developed.

Advancing manufacturing

Tissue-engineered medical products (TEMPs) represent a burgeoning industry, one that has drawn much attention to cell and gene therapies. Yet TEMPS, insists Tom Bollenbach, PhD, chief technology officer of the Advanced Regenerative Manufacturing Institute (ARMI), should also stimulate a reexamination of manufacturing issues.

At ARMI, a Manchester, NH-based nonprofit organization, the mission is to make consistent and cost-effective manufacturing practical by producing modular and scalable GMP-compliant processes and integrated technologies, and by developing standardized manufacturing practices. Programs are supported by awards, member-matching contributions, membership fees, and other grants and contracts.

In December 2016, the Department of Defense awarded ARMI $80M to operate BioFabUSA, which launched mid-2017. BioFabUSA is a 150+ member, public-private partnership comprising companies, academic institutions, and not-for-profit organizations in the TEMP ecosystem. Members are eligible for funding. To date, over $33.3M has been approved for TEMP manufacturing projects. BioFabConsulting, a premier regulatory consulting firm for complex TEMPs, supports members.

Advanced Regenerative Manufacturing Institute
With support from the U.S. Department of Defense, the Advanced Regenerative Manufacturing Institute operates BioFabUSA, a program that is developing the Tissue Foundry, a modular manufacturing system. It integrates multiple platform technologies and can be reconfigured to produce varied Tissue Engineered Medical Products (TEMPs). Ultimately, it will be validated to allow GMP-compliant manufacturing.

BioFabUSA has identified capability gaps preventing scalable, consistent, and cost-effective manufacturing. These include the need for a robust supply chain of more consistent raw materials, increased automation, more appropriately designed equipment, better measurement and data management tools, methods for the preservation and transport of cells and tissues, and scalable manufacturing processes and methodologies.

To close these capability gaps and help achieve BioFabUSA’s goals, membership teams are collaborating on the development of the modular Tissue Foundry manufacturing system. The Tissue Foundry, which integrates many developing platform technologies, can be reconfigured to produce any TEMP, and, ultimately, will be validated to allow GMP-compliant manufacturing.

“In the past, TEMP manufacturing seemed too complicated and too costly,” says Bollenbach. “Manufacturing improvements, new tools, measurement technologies, and manufacturing data will drive the cost reductions needed to attract investors and more widely advance the field.”

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