Clinical Variables that Influence Properties of Human Mesenchymal Stromal Cells Abstract Many bone tissue engineering studies use animal or immortalized human mesenchymal stromal cells (hMSCs), which include osteoblast progenitors, because of the abundance of those cells. It is advantageous to study the less plentiful human MSCs in research on bone tissue engineering because of the clinical relevance and because of the importance of discovering how to optimize hMSCs for autogenous use. There is growing evidence that some of the irreproducibility in biological experiments with hMSCs can be attributed to variable clinical characteristics of the subjects from whom they were obtained. Patients who could benefit from bone tissue engineering are likely to have cellular deficits and compromised biochemical milieu, some of which can be managed by customized in vitro treatments. Many donor characteristics are associated with their hMSC in vitro properties. Studies with cohorts with similar characteristics show that some deficits are modifiable in vitro and can be managed with greater understanding of their pathophysiological mechanisms. Even hMSCs from elders can be rejuvenated in vitro with safe agents. Additional value in studying physiology of hMSCs from characterized subjects is to develop rationales for new in vivo therapies to ensure skeletal health throughout the lifespan. Lay Summary During this era of intensive tissue engineering research, it is appealing to use human mesenchymal stromal cells (hMSCs) for bone tissue engineering research because of their clinical relevance. Use of the patient’s own cells for therapeutic applications is called autogenous cell-based therapy. Research shows that properties of hMSCs isolated from a subject’s marrow depend on many clinical characteristics and that, in some cases, their osteoblast differentiation potential in cell culture can be optimized safely. These observations also suggest ways to optimize the functions of the skeleton throughout the lifespan.

The Background and Scope of Polyphosphazenes as Biomedical Materials Abstract Although many traditional organic polymers have been evaluated for uses in biology and medicine, relatively few have proved to be satisfactory for crucial uses such as surgical sutures or mesh, tissue engineering substrates, controlled drug delivery, or soft matter. A reason for this is that most large-volume commercial polymers are optimized for some mechanical engineering purpose, and biomedical compatibility or bioerodibility is not one of the target properties. Thus, biomedical scientists and engineers have been forced to improvise and compromise by using widely available non-biological polymers, mainly because those materials are already available in commercial quantities. Polyphosphazenes offer an opportunity to solve many of these problems. Lay Summary A crucial need exists for new polymers that can be utilized in orthopedics, cardiovascular, dental, neural, or drug delivery applications, yet very few long-existing polymers have properties that are ideal for these medical uses. Our research seeks to design and find methods to synthesize polymeric materials that are specifically designed to solve a range of medical challenges. In our program, we make use of an unusual polymer backbone comprised of alternating phosphorus and nitrogen atoms to which are attached a wide range of organic side groups. This system is unique in the wide range of different property combinations that can be generated, and many of these combinations can be matched precisely to the needs of medical materials.

Identifying and Managing Sources of Variability in Cell Therapy Manufacturing and Clinical Trials Abstract Identifying and managing cell therapy variability can be a significant challenge for a company seeking to commercialize a new product. Failure to address this issue can lead to negative consequences such as delayed approval due to unsuccessful clinical investigations, or failed product lots that do not meet release criteria. Allogeneic cell therapies can be particularly prone to variability challenges due to the use of variable input material. In order to support the manufacturers of cell therapies, the FDA has identified two primary regulatory pathways (351 vs 361) that reflect the relative risk of the product. In this review, we will discuss criteria that separate the two potential regulatory pathways for cell therapy products in the USA. Also, we will discuss what aspects of manufacturing and clinical trial execution might introduce undesired variability that can derail the path towards licensure and commercialization, along with tools to minimize these potential sources of variability. ClinicalTrials.gov Identifier: NCT03347708 and NCT03955315 Lay Summary Therapies that utilize live cells as the active ingredient, known as cell therapies, are a promising approach to treating many diseases that cannot be addressed with traditional medicines. However, with this great potential comes specific challenges associated with cell therapy, including identifying the appropriate regulatory approval pathway, manufacturing it in a reproducible way, and successfully executing clinical trials. In this review, we describe potential sources of variability that can negatively impact the translation of a cell therapy, and ways to minimize those risks.

News and Views September 2019

Altered Biodistribution and Tissue Retention of Nanoparticles Targeted with P-Glycoprotein Substrates Abstract Low molecular weight substrates of the efflux transporter, P-glycoprotein, alter the biodistribution and tissue retention of nanoparticles following intravenous administration. Of particular interest is the retention of the targeted nanoparticles in the brain. Drug delivery to the brain is hindered by the restricted transport of drugs through the blood-brain barrier (BBB). Drugs that passively diffuse across the BBB also have large volumes of distribution; therefore, alteration of their biodistribution to increase their concentration in the brain may help to enhance efficacy and reduce off-target side effects. In this work, targeted nanoparticles were used to explore a new approach to target drugs to the brain—the exploitation of the P-glycoprotein efflux pump. The retention of nanoparticles containing a strong P-glycoprotein substrate, rhodamine 6G, tethered to a PLA nanoparticle through a PEG spacer was greater than twofold relative to untargeted nanoparticles and to nanoparticles tethered to a weaker P-glycoprotein substrate, rhodamine 123. In a P-glycoprotein knockout mouse model (mdr1a (-/-)), there were no significant differences in brain accumulation between rhodamine 6G-targeted particles and controls, strongly supporting the role of P-glycoprotein. This proof of concept report shows the potential applicability of low molecular weight P-gp substrates to alter nanoparticle biodistribution. Lay Summary The efficacy of medicines can be improved by diverting drugs to specific tissues. Finding new ways to target medicines to diseased tissue is an active area of research across disciplines. Drug-loaded nanoparticles, delivered to tissues of interest, are one way to accomplish this goal. The work reported in this manuscript explores the possibility of using small molecules to get nanoparticles to bind to a drug efflux pump, P-glycoprotein (P-gp), that is present in various tissues in the body. P-gp functions to remove drugs from tissues, and it is usually considered a hindrance to drug targeting. The research in this paper shows that the natural function of P-gp can be used favorably to retain nanoparticles in various tissues. Future Work The data reported in this manuscript serves to establish a proof-of-concept that low molecular weight P-gp substrates can be used to alter the biodistribution of nanoparticles. Future work includes (1) understanding the targeting mechanism(s) that lead to these results, (2) identifying FDA-approved drugs that can target nanoparticles, and (3) evaluating how nanoparticle biodistribution is altered by using P-gp substrates with different binding constants.

Skeletal Muscle Regenerative Engineering Abstract Skeletal muscles have the intrinsic ability to regenerate after minor injury, but under certain circumstances such as severe trauma from accidents, chronic diseases, or battlefield injuries the regeneration process is limited. Skeletal muscle regenerative engineering has emerged as a promising approach to address this clinical issue. The regenerative engineering approach involves the convergence of advanced materials science, stem cell science, physical forces, insights from developmental biology, and clinical translation. This article reviews recent studies showing the potential of the convergences of technologies involving biomaterials, stem cells, and bioactive factors in concert with clinical translation, in promoting skeletal muscle regeneration. Several types of biomaterials such as electrospun nanofibers, hydrogels, patterned scaffolds, decellularized tissues, and conductive matrices are being investigated. Detailed discussions are given on how these biomaterials can interact with cells and modulate their behavior through physical, chemical, and mechanical cues. In addition, the application of physical forces such as mechanical and electrical stimulation is reviewed as strategies that can further enhance muscle contractility and functionality. The review also discusses established animal models to evaluate regeneration in two clinically relevant muscle injuries: volumetric muscle loss (VML) and muscle atrophy upon rotator cuff injury. Regenerative engineering approaches using advanced biomaterials, cells, and physical forces, developmental cues along with insights from immunology, genetics, and other aspects of clinical translation hold significant potential to develop promising strategies to support skeletal muscle regeneration. Lay Summary Skeletal muscle has robust regeneration properties, but in extreme conditions, the regeneration ability is hindered. It remains a common clinical problem that could lead to long-term disability. The available treatments such as muscle flap transposition present various limitations. To address these limitations, promising strategies based on regenerative engineering are being developed. This review article discusses the different approaches to tissue regeneration using the regenerative engineering paradigm. A specific discussion involves biomaterials and their interactions with cells and bioactive molecules. In addition, the advantages of physical and mechanical stimulation in muscle regeneration are discussed.

Regulation of Cardiomyocyte Differentiation, Angiogenesis, and Inflammation by the Delta Opioid Signaling in Human Mesenchymal Stem Cells Abstract Background Lack of angiogenesis, inflammation, and low differentiation of MSCs upon transplantation to ischemic site are major drawbacks in cell therapies for myocardial tissue regeneration. To overcome these problems, this study aimed to increase the angiogenesis, anti-inflammation, and differentiation of human umbilical cord MSCs (hMSCs) into cardiomyocytes by activating the delta opioid pathway by its agonist SNC80 with the combination of zebularine, activin A and oxytocin. Materials and Methods Human umbilical cord MSCs were seeded on gelatin-coated plates by using alpha MEM. MSCs were treated with SNC80 and the combination of zebularine, activin A and oxytocin. Media and inducers were changed every 3 days once until 21 days. Spent media collected from day 7 and day 21 was used to estimate angiogenic (VEGF) and anti- or pro-inflammatory cytokines (IL-10, IL1b, IL-6) levels by ELISA. The effect of spent media on in vitro endothelial tube formation and anti-inflammatory effect on inflammation induced with LPS on macrophages was also studied along with cytokine levels. Proteins that were isolated from day 7 and day 21 were used to study for cardiomyocyte markers (GATA4, MEF2C, connexin 43, and KCNJ2) and NOTCH1 involved in cardiomyocyte differentiation using Western blot. Results Activation of DOR increases the VEGF secretion levels estimated by ELISA and increases the in vitro tube formation in terms of number of loops and total length. Delta opioid receptor (DOR)-activated conditioned media secreted anti-inflammatory cytokine IL-10, which could prevent the secretion of pro-inflammatory cytokines IL-1b and IL-6 from LPS induced macrophages. DOR activated MSCs upregulated the early and late cardiomyocytes markers (GATA4, MEF2C and connexin 43, KCNJ43) via NOTCH1 signaling. Conclusion Activation of DOR on hMSCs plays a vital role in angiogenesis, anti-inflammation, and cardiomyocyte differentiation by regulating the NOTCH1 signaling pathway. Lay Summary Our results indicate that, upon activation of delta opioid receptors on human umbilical cord derived mesenchymal stem cells, they can differentiate into heart cells (by expressing specific markers GATA4, MEF2C, Connexin43), show enhanced formation of blood vessels and anti-inflammatory activity. This approach may help to treat pateints post heart attack.

Optimizing 3D Co-culture Models to Enhance Synergy Between Adipose-Derived Stem Cells and Chondrocytes for Cartilage Tissue Regeneration Abstract Donor cell scarcity is a key barrier for clinical translation of chondrocytes for regenerating cartilage. To overcome this challenge, recent studies have utilized mixed populations of chondrocytes and adult stem cells to show synergistic interactions during 3D co-culture using hydrogels or pellets. Yet, how different co-culture models impact the synergy and resulting cartilage formation remains unknown. To determine the optimal delivery method of a mixed population of stem cells and chondrocytes for cartilage regeneration, here we compared hydrogel and pellet co-culture on the interactions between adipose-derived stem cells (ADSCs) and neonatal chondrocytes (NChons). While both co-culture models supported synergy, hydrogel co-culture led to over a 5-fold increase in synergistic index in cell proliferation, and over 11 and 15-fold increases in synergistic indices for production of cartilage matrix (collagen and sGAG respectively). Interaction index analyses of gene expressions showed hydrogel co-culture significantly reduced hypertrophic phenotype of both ADSCs and NChons. In hydrogel co-culture, NChons contributed to the neocartilage deposition while ADSCs resided outside the newly formed cartilage nodule, serving as supporting cells through paracrine signaling. In contrast, during pellet co-culture, both NChons and ADSCs contributed to the newly formed cartilage. Using the same number of cells, hydrogel co-culture supported higher synergy and produced neocartilage tissues approximately 5 times the size of pellet co-culture. In contrast, pellet co-culture resulted in denser cartilage matrix suitable only for small defects. The outcomes of this study suggest hydrogel delivery as a more advantageous co-culture method for regenerating cartilage using mixed cell populations. Lay Summary Donor cell scarcity is a key barrier for clinical translation of cell-based therapies for cartilage regeneration. To overcome this challenge, here we harnessed abundantly available fat-derived stem cells and mixed them with juvenile chondrocytes, a cell type from native cartilage that can produce robust cartilage. We compared two different co-culture methods, hydrogel versus cell pellet, to grow the mixed cell populations for cartilage regeneration. We demonstrate that 3D hydrogels induced higher synergy using mixed cell populations, allowing cartilage repair with a reduced number of chondrocytes and defect filling of larger volumes than pellets. Future Works Future work will validate the potential of using hydrogels to deliver mixed population of fat-derived stem cells and juvenile chondrocytes for cartilage regeneration in vivo using relevant animal models.

Silicon-Substituted Hydroxyapatite Particle s and Response of Adipose Stem Cells In Vitro Abstract Due to the similarity of synthetic hydroxyapatite (HA) to natural bone tissue, but, because of its low degradation rate, the current study focuses on silicon-substituted HA (Si-HA) synthesis, characterization, and biological evaluations. Si-HA was successfully prepared through sol-gel processing route and characterized using SEM, EDX, XRD, and FTIR. Si-HA particles were found to be non-cytotoxic following exposure to adipose stem cells (ADSCs). In fact, Si-HA particles showed a high level of matrix mineralization following prolonged and continuous exposure to ADSCs. It is suggested that the incorporation of Si in HA structure positively affects cellular behavior, associated with a higher degradation rate, and subsequently greater level of ionic product release from Si-HA particles. Lay Summary Hydroxyapatite (HA) has long been applied as bone substitutes but its low degradation rate limits its application. One approach is the incorporation of silicon (Si) within HA structure. This study confirms that Si-substituted HA enhance stem cell proliferation and promote osteogenic differentiation. Hence, Si-HA could be utilized in composites, scaffolds, and coatings for bone-related disorders.

Monocyte Modulation by Liposomal Alendronate Improves Cardiac Healing in a Rat Model of Myocardial Infarction Abstract The ischemic injury in acute myocardial infarction (AMI) activates the innate immunity response in two consecutive phases. Classical monocytes (CM) accumulate in the inflammatory phase (first 3 days), and non-classical monocytes (NCM) accumulate in the reparatory phase (4–7 days). We hypothesized that inhibition of monocytes at the second phase post-AMI will lead to better healing by reducing myocardial damage and consequently improve heart function. We examined the effect of monocyte modulation on cardiac healing following MI injury in rats by nano-sized alendronate liposomes (LipAln) treatment. Rats were treated with intravenous (IV) LipAln, on days 5, 7, and 9 after ligation of the left anterior descending artery (LAD). Circulating monocyte levels were reduced after the first LipAln injection, and two peripheral blood monocyte subsets, CM and NCM, were sequentially mobilized after MI. Two weeks after MI, a reduction in infarct size was observed and cardiac function was improved in LipAln-treated rats (fractional shortening of 32.2% ± 1.9% and 26.0% ± 1.3%, for LipAln and saline treated rats, respectively, p < 0.05). This improvement was further corroborated by increased cardiac anti-inflammatory cytokine expression and reduced levels of pro-inflammatory cytokines. In conclusion, LipAln treatment during the second phase after MI improves cardiac healing. Lay Abstract Myocardial infarction (MI) occurs when coronary blood flow decreases, causing damage to the heart muscle. Consequently, the body sends cells called monocytes/macrophages as part of a reparatory-inflammatory response. We hypothesized that altering a specific step in the inflammatory process, which the heart utilizes to heal itself, could result in improved heart function. Using a unique drug delivery system of nano-sized particles called liposomes, in which a molecule (bisphosphonate) that is toxic to monocytes is embedded, we successfully altered the inflammatory process, in a rat model of MI. resulting in improved heart function. The novel technology reported in this issue celebrating Robert Langer’s birthday is directly linked to my previous work with Bob. As Bob’s postdoc at MIT (1984–1986), our group developed heart valve anti-calcification implantable drug delivery systems, which contain a bisphosphonate. His guidance and mentorship then, and to this day, are of great importance. I am forever grateful for his continued contribution. Graphical Abstract