Laboratory for Therapeutic 3D Bioprinting

Lab Director Brian Grottkau, MD, discusses the Lab’s latest research and clinical goals.

Tissue and organ injury or dysfunction is a feature of many diseases. Cell therapy has been used to treat some of these diseases. Some cell therapeutics are currently approved for use by the FDA, while others are in clinical trial. A roadblock for many of these therapies is that these cells often have suboptimal function and viability when delivered in suspension because they lose their micro-environment. Tissue engineering approaches can improve cell function by combining cells, scaffold and biomolecules together to recreate a functional 3-dimensional tissue.

Bioprinting is an emerging technology that can spatially control the construction process of an engineered tissue.

While most current 3D bioprinting technologies focus on the anatomical shape of tissues, The Laboratory for Therapeutic 3D Bioprinting at Mass General focuses on controlling the architecture of tissues at the microscopic level. Our mission is to develop submillimeter-sized micro-tissues with specific morphology, biochemical and biomechanical functions that are comparable to human tissues using our patent-protected 3D bioprinter. We can deliver these tissues to an injured site in the body in a minimally invasive manner, where they self-assemble into macro-sized tissues to repair the injured tissue or organ.

Our lab is also developing a novel approach to personalized cancer care. Using our proprietary 3D printer, we can reproduce a patient’s tumor using the patient’s own cancer cells, cancer-related cells and matrix. We can test these tumors using high-throughput technology against individual or combinations of clinically available chemotherapeutic agents. The results may help oncologists make better informed treatment decisions.

Our 3D bioprinted micro-tissues can be used in the lab for:

• Studying tissue and organ regeneration
• Exploring mechanisms of diseases
• Drug sensitivity testing
• Drug development

We are developing broad collaborations with various stakeholders to investigate other potential avenues of investigation and treatment.

Research Projects

Articular cartilage regeneration using bioprinted micro-cartilage

Osteoarthritis and articular cartilage lesions affect tens of millions of Americans each year and are the leading cause of disability in the US. Articular cartilage does not have the capacity to heal on its own and current treatment options for articular cartilage lesions have varied drawbacks. Our laboratory has successfully bioprinted micro-articular cartilage using our innovative 3D bioprinting system and implanted the cartilage into defects through a minimally invasive approach.

Our micro-articular cartilage tissue has been shown to completely cure large articular cartilage defects that approximate those clinically observed in humans. Our bioprinted micro-articular-cartilage demonstrates standard morphology and biochemical, biomechanical and molecular profiles that are comparable to native articular cartilage. Our micro-cartilage can be implanted arthroscopically and starts to self-assemble hours after implantation. The cartilage then continues to grow and mature in vivo.

The implanted micro-cartilage also effectively incorporates with the surrounding native cartilage and subchondral bone and remains in place even in the presence of axial, rotatory and shear forces. Histology and immunohistology results demonstrate good integration of the bioprinted cartilage into the surrounding cartilage and subchondral bone.

We aim to begin clinical trials within 2 years.

Bioprinting micro-tumor tissues for personalized cancer therapy

25 percent of all deaths in the US resulted from cancer in 2014. Cancer therapy is transitioning to precision medicine, which aims to identify the best treatment regimen for each individual patient. Gene sequencing and functional drug testing are two approaches currently being utilized for precision medicine. Unfortunately, there are two major limitations to gene sequencing for drug sensitivity prediction. First, it is an indirect approach because the drug response prediction is based upon the drug response of other patients with the same mutation. Second, the correlation between the mutations and drug sensitivity are largely unknown and there are a limited number of biomarkers that can be used to predict the drug sensitivity.

Functional drug testing is a more direct approach that is performed to assess the sensitivity of available drugs against an individual patient’s own cancer cells. Currently, 2-dimensional and simple 3-dimensional culture models are being utilized and they are often unable to recreate the tumor microenvironment, which leads to poor predictions of in vivo drug responses. A PDX mouse model take a long time to generate than is clinically advisable for the initiation of definitive therapy. Usually, not enough animals are generated to be used for drug screening. Additionally, the mouse environment often changes the genotype and phenotype of the implanted xenograft, which makes it less useful for drug sensitivity testing. It is critical to have a reliable way to predict patient response to medications to select the best treatment plan.

Utilizes our 3D micro-tissue bioprinting technology, we have developed 3D micro-cancer models that recreate the tumor micro-environment (including cellular and extracellular matrices). Our bioprinted micro-cancer models have the following advantages over existing models:

1. Recreated cellular components including primary cancer cells, cancer associated fibroblasts and microvascular networks
2. Recreated extracellular matrix
3. Recreated chemical micro-environment including hypoxia gradient
4. Recreated physical barrier micro-environment

We have found that our 3D bioprinted tumors demonstrate the following characteristics: High viability during the relatively long-term culture, morphology comparable to the native tumors, similar proliferation patterns to in vivo tumors, invasive behavior mimicking native tumors, pro-angiogenesis behavior, and more drug resistance. When grafting, the rate and degree of tumorigenicity were improved compared to conventional grafting methods.

Our 3D bioprinted tumor models provide a versatile platform for cancer biology study, cancer drug development and selecting specific drug based on the tumor’s sensitivity for an individual patient.

3D bioprinting vascularized micro liver tissue for live regeneration and drug metabolism study

Liver disease is the twelfth most frequent cause of death for people of all ages and fourth most frequent for the middle-aged in the US. Over 15,000 people are on the liver transplantation waiting list. An average of 1,500 people die each year while waiting for a liver transplant. To overcome the liver donor shortage, great efforts have been made on hepatocyte transplantation to treat liver damage, which is less invasive than orthotopic liver transplantation. However, hepatocyte transplantation faces the challenges as all cell transplantation therapies--low survival rates and impaired functions.

Our lab bioprints micro liver tissues that are delivered to the location of the damaged liver through a minimally invasive approach. Because hepatocytes consume much more oxygen than other types of cells, vascularizing an engineered tissue is important. We have successfully bioprinted vascularized micro liver tissues, which significantly improved their viability and function. The bioprinted micro liver can also be applied to drug metabolism studies, which are critical in drug development.

Engineering anatomically shaped bone using 3D printing

Anatomically shaped scaffolds are critical in generating implantable engineered tissue. Solvent casting/particulate leaching is a widely-used technique to generate porous scaffolds. However, generating anatomically shaped casting molds is challenging because of the manufacturing limitations of conventional molding materials such as ceramic and Teflon. Additionally, solvent and salt removal takes days.

To solve these problems, our lab has developed a novel technique utilizing 3D printing of the mold with rapid removal of the solvent and particulates by perfusion. We utilize clinical CT images of a human femur to generate a 3D digital model. The model has identical geometry to the human femur with add-on ports for solvent casting and perfusion. The anatomically shaped mold was 3D printed using a solvent-resistant and water-soluble polymer. A bone inductive scaffold was developed with hydroxyapatite (HA) and PCL- or PLA-polymer using the solvent casting/particulate leaching method in the printed mold.

Compared with conventional methods, this perfusion technique completely removed the solvent by ethanol perfusion within several minutes and removed particulates by water perfusion in a 3D printed none-water soluble mold within a few hours. The scaffold shows high porosity and interconnectivity. The scaffold also shows good biocompatibility as evidenced by seeding osteoblastic cells, which demonstrated good viability, proliferative activity and osteogenic differentiation. Our technology is a versatile tool to efficiently create patient-specific bone grafts. It is also applicable for many other biomaterials and many other tissues.

3D bioprinting angiogenesis model

Angiogenesis is fundamental to organ formation, wound healing, tumor growth, invasion and metastatic dissemination. 3D angiogenesis models better mimic in vivo conditions than 2D models. However, conventional angiogenesis models don’t represent the complexity in the micro environment during angiogenesis.

Using our 3D bioprinting technology, we have successfully generated complex 3D angiogenesis models, which better resemble the in vivo conditions. In addition, the customized printing process enable the 3D model to facilitate high-throughput screening with multiple conditions.

Collective cell migration model generated by 3D printing

Collective cell migration (cells migrate as a group) is fundamental in many biological and pathological processes. However, existing methods have drawbacks such as cell damage, substrate surface alteration, limitation in media exchange and solvent interference. The superhydrophobic surface has a water contact angle of greater than 150 degrees.

Our lab has successfully developed a patterned superhydrophobic array for studying collective cell migration in high-throughput. The array was developed on a rectangular single-well cell culture plate consisting of hydrophilic flat microwells separated by the superhydrophobic surface. The main manufacturing process includes patterning discrete protective masks to the substrate using 3D printing, robotic spray coating of silica nanoparticles, robotic mask removal, robotic mini silicone blocker patterning, automatic cell seeding and liquid handling.

Compared with a standard 96 well plate, our system increases the throughput by 2.25 fold and non-destructively generates a cell-free area in each well. Our system also demonstrates high-efficiency in liquid handling compared with conventional microwell plates. The superhydrophobic surface had no negative impact on cell viability. We studied the collective migration of human umbilical vein endothelial cells and cancer cells using assays of endpoint quantification, dynamics cell tracking and migration quantification following varied drug treatments. This system provides a versatile platform to study collective cell migration in high-throughput for a broad range of applications.

Research Positions

The Laboratory for Therapeutic 3D Bioprinting teams with individuals with diverse backgrounds and establishes collaborations with groups with diverse research interests. We are looking for highly motivated and dedicated individuals who are passionate about advancing 3D bioprinting technology for therapeutic purposes. We welcome candidates with medical, biological, pharmaceutical and engineering backgrounds.

Interested individuals seeking positions as research fellows, international exchange scholars/students or student interns, should forward the statement of interest, CV and three references to Dr. Yonggang Pang.

Selected Publications

Yonggang Pang, Jing Yang, Zhixin Hui, Brian E. Grottkau. Robotic Patterning a Superhydrophobic Surface for Collective Cell Migration Screening. Tissue Engineering Part C, 2018.

Yang, B. Grottkau, Z. He, C. Ye. Three-dimensional printing technology and materials for treatment of elbow fractures. Int Orthop. 2017, pp. 2381-2387.

Luo, K.M. Kulig, E.B. Finkelstein, M.F. Nicholson, X.H. Liu, S.M. Goldman, J.P. Vacanti, B.E. Grottkau, I. Pomerantseva, C.A. Sundback, C.M. Neville. In vitro evaluation of decellularized ECM-derived surgical scaffold biomaterials. J Biomed Mater Res B Appl Biomater. 105 (2017) 585-593.

Zhou, B.E. Grottkau, S. Zou. Regulators of Stem Cells Proliferation in Tissue Regeneration. Curr Stem Cell Res Ther. 11 (2016) 177-187.

BE Grottkau, Z Hui, SK Saha, NM Bardeesy, Y Pang. 3d Bioprinting Cancer Tissues for Drug Development and Personalized Cancer Therapy. Tissue Engineering Part A. 22 (2016) S134-S135.

Z Hui, D Ma, Y Pang, B Grottkau. Bioprint Injectable MSC Microtissues Repairing Cartilage Defects. Tissue Engineering Part A. 22 (2016) S83-S83.