EVERY QUARTER NIDCR INVITES AN EXPERT, AND WE ARE VERY PLEASED TODAY TO HAVE DR. DAVID MOONEY. HIS WORK ACTUALLY HIGHLIGHTS ONE OF THE FIVE NIDCR 2030 STRATEGIC INITIATIVES, THAT'S AUTOTHERAPIES, INNOVATIVE THERAPIES THAT ACTUALLY HARNESS THE BODY'S INNATE ABILITY TO EITHER HEAL OR REPAIR OR FIGHT DISEASE. AND DR. MOONEY'S RESEARCH ACTUALLY HAS A NUMBER OF APPLICATIONS. ONE IN PARTICULAR THAT I SAW WAS PATHOLOGIC RELEVANCE OF MATRIX STIFFNESS AND AHOW THAT AFFECTS KINNETT KINETIC GROWTH PATTERNS IN MYELOID LEUKEMIA. HE'S AN ENGINEER, ABLE TO MARRY TWO DISCIPLINES IN A BEAUTIFUL WAY. SO, JUST A LITTLE BIT ABOUT DAVID MOONY. HE RECEIVED HIS Ph.D. AT M.I.T. HE IS A BONA FIDE ENGINEER. YET WHEN YOU HEAR HIM SPEAK SOMETIMES YOU FORGET AND THINK HE MUST BE A PHYSICIAN. HE SPENT THIS LAST -- THE NEXT 10 YEARS AT THE UNIVERSITY OF MICHIGAN, AND RETURNED TO HARVARD TO BE THE ROBERT PINKAS FAMILY IN BIOENGINEERING AT HARVARD, CORE MEMBER OF THE WYSS INSTITUTE. WHAT STOOD OUT IN HIS C.V. IS THE FACT HE'S NOT -- HE HASN'T RECEIVED JUST ONE AWARD, NOT JUST TWO, BUT THREE AWARDS IN MENTORING EXCELLENCE. AND BECAUSE OUR NIDCR GRAND ROUNDS IS DEDICATED TO OUR CLINICAL RESEARCH FELLOWS, I THINK THAT WAS VERY APPROPRIATE. WE'VE GOT A WONDERFUL CHOICE IN TERMS OF OUR SPEAKER TODAY. PLEASE BE PREPARED TO HAVE YOUR IMAGINATION STRETCHED. SO PLEASE JOIN ME IN WELCOMING DR. DAVID MOONEY. [APPLAUSE] >> SO THANK YOU SO MUCH FOR THE OPPORTUNITY TO COME AND ENGAGE WITH THE COMMUNITY HERE. I ALWAYS GREATLY ENJOY COMING TO THE NIH, PARTICULARLY THE NIDCR. A LOT OF EXCITING RESERVE, I'M LOOKING FORWARD TO A DAY OF STIMULATING CONVERSATIONS. I'M GOING TO TALK TODAY ABOUT SOME WORK WE'RE DOING IN THE LAB THAT RELATES TO HOW MECHANICS MAY PLAY A ROLE IN REGENERATION, AND THE WAY I'LL ORGANIZE MY TALK, WELL FIRST, I WANT TO ACKNOWLEDGE A GREAT NUMBER OF PEOPLE THAT I'VE COLLABORATED WITH IN THE WORK I'LL DESCRIBE. ALL THE RESEARCH I'LL DESCRIBE TODAY, WE GET FUNDING FROM MANY PLACES IN THE LAB, WHAT I DESCRIBE TODAY IS HAS BEEN FUNDED BY THE NIDCR, MAYBE TWO OR THREE SLIDES OF DATA FUNDED ELSEWHERE, OTHERWISE IT'S ALL NIDCR FUNDING THAT'S ENABLING THIS REALLY EXCITING OR I THINK EXCITING RESEARCH THAT WE'RE DOING. AND THE STUDENTS AND POSTDOCS WHO DID THE WORK I'LL ACKNOWLEDGE AS I GO THROUGH. I DECIDED TO TALK TODAY AND FOCUS ON RULE OF MECHANICS. IT'S SOMETHING THAT IN DENTISTRY IS INTUITIVELY OBVIOUS TO EVERYONE IN THE ROOM. ORTHODONTICS,, OSTEOGENESIS, MANY HAVE BEEN TRYING TO UNDERSTAND SIGNALING PATHWAYS AS YOU SEE IN THE BOTTOM OF THE SLIDE. I'M NOT GOING IN DETAIL IN TERMS OF SIGNALING PATHWAYS TODAY. INSTEAD I'M GOING TO TALK ABOUT HOW WE CAN USE MECHANICAL CUES IN THE CONTEXT OF REGENERATION, THAT'S THE FIRST 75% OF MY TALK. THE LAST PART, A CLINICAL COMPONENT TO THE AUDIENCE TODAY, I WANT TO DESCRIBE HOW THAT BASIC AND FUNDAMENTAL RESEARCH LED US INTO AN UNEXPECTED AREA, THIS OFTENTIMES HAPPENS IN RESEARCH IF WE KEEP OUR EYES OPEN, WE FIND THINGS WE DON'T EXPECT. THIS LED TO US DEVELOP A NEW TYPE OF MEDICAL ADHESIVE, THIS IS JUST A VIDEO SHOWING THIS ADHESIVE BEING PUT ON A BEATING PIG HEART, WE PUT SOME BLOOD DOWN TO MIMIC A BLOODY SURGICAL FIELD. WHAT YOU'LL SEE THIS ADHESIVE WE'RE PLACING ONTO THE HEART AND WILL HOLD IT IN PLACE, ABOUT A MINUTE, APPLY COMPRESSION AND WE'LL BASICALLY TAKE THIS BACKING OFF, AND NOW WE'LL TRY TO PULL OFF THE ADHESIVE. WHAT YOU SEE, WE DO THIS REALLY PRIMITIVE PEELING TEST IN VIVO, IS THE ADHESIVE WAS BONDED HERE AND NOW WE'RE PULLING IT OFF, YOU CAN GET A SENSE OF HOW WELLED WELLED ADHERENT THIS WAS TO BEATING HEART IN A BLOODY ENVIRONMENT. THIS WAS THE INSPIRATION FOR THE DEVELOPMENT OF THIS. I'LL DESCRIBE THAT IN DETAIL AS I GO ALONG. NOW BACK TO THE BEGINNING. THE BEGINNING FOR US WE'VE HAD A LONGSTANDING INTEREST IN THE IDEA CELLS MAY BE USEFUL THERAPEUTIC AGENTS, IN PARTICULAR WE'VE BEEN INTERESTED IN THE CONCEPT OF USING STEM CELLS, NOT A NEW PREMISE FOR US, THE FIELD HAS BEEN INTERESTED IN THIS FOR 20, 25 YEARS NOW. THERE'S TISSUE-ENGINEERED SKIN PRODUCTS USED TO TREAT PATIENTS, THERE'S THAT MIDDLE IMAGE THAT MANY PEOPLE IN THE AUDIENCE ARE QUITE FAMILIAR WITH, A CASE IN GERMANY THE MANDIBLE WAS ENGINEERED, USING COMBINATION OF CELLS AND MORPHOGENS AND MATERIALS. ON THE FAR RIGHT ONGOING CLINICAL WORK FROM CHRIS BREWERS'S LABORATORY, CONGENITAL DEFECTS IN CHILDREN ENGINEERING BLOOD VESSELS. A CHALLENGE WITH STEM CELLS AS YOU CAN SEE IS HOW TO TAKE THE CELLS AND GET THEM TO FORM STRUCTURES WITH DEFINED FUNCTIONALITIES WE NEED FOR PARTICULAR APPLICATION. THE APPROACH WE TAKE, THAT MANY GROUPS TAKE IN THIS FIELD, IS TO GO BACK TO THE NORMAL ENVIRONMENT WHICH THE STEM CELLS LIVE, STEM CELL NICHE, TO TRY TO UNDERSTAND AND THEN EXPLOIT SOME OF THE NORMAL SIGNALS THAT THESE CELLS RECEIVE IN THEIR NICHE AND BUILD THESE INTO THE SYSTEMS THAT WE ENGINEER. IN PARTICULAR IN MY LABORATORY, WE FOCUS ON THE IDEA OF USING BIOMATERIALS AS YOU SEE A FEW EXAMPLES HERE, IN WHICH WE CAN PRESENT AND ORGANIZE THESE DIFFERENT KINDS OF CUES TO CONTROL STEM CELL BEHAVIOR. I'M NOT GOING TO TALK ABOUT ALL THE CUES BUT FOCUS ON ONE, MECHANICAL CUES. THE REASON IS PRETTY OBVIOUS, WHICH THIS VIDEO DEMONSTRATES NICELY, THIS IS A HUMAN CELL, MIGRATING THROUGH A COLLAGEN SCAFFOLD. WHAT YOU CAN APPRECIATE, AS THE WELL MOVES THROUGH THE SCAFFOLD IT GRABS AND PULLS THE COLLAGEN FIBRILS AND MOVES THEM AROUND. WHAT THIS IS A NICE DEMONSTRATION IS THAT CELL ADHESION AND CELL INTERACTION WITH MATERIALS IS A MECHANICAL EVENT, AS WELL AS CHEMICAL EVENT. AND OF COURSE THIS THEN LED TO THE QUESTION, WELL, IF THE EXTRACELLULAR MATRIX RESISTS FORCES FROM CELLS TO CELLS RESPOND TO THE RESISTANCE THEY FEEL FROM THE ENVIRONMENT? DOES THIS STIFFNESS OF THE SUBSTRATE ALTER HOW A CELL RESPONDS IN A PARTICULAR MICROENVIRONMENT? NOW, THIS IS SOMETHING THAT'S BEEN OF INTEREST AND WIDELY EXPLORED IN TWO DIMENSIONAL CELL CULTURE OVER THE LAST 20 YEARS, PERHAPS THE FIRST NICE EXAMPLE WAS FROM WANG'S LAB WHERE HE SHOWED CELL MIGRATION SPEED WAS REGULATED BY STIFFNESS OF THE SUBSTRATE, CELLS WERE ATTACHED TO. WE SHOWED THINGS LIKE PROLIFERATION, APOPTOSIS OF STEM CELLS WAS REGULATED BY STIFFNESS OF THE SUBSTRATE, AND DENNIS SHOWED SEVERAL YEARS AGO THE DIFFERENTIATION OF MESENCHYMAL STEM CELLS DOWN DIFFERENT PATHWAYS WAS REGULATED BY STIFFNESS OF THE CUB -- SUBSTRATE. THE WORK WAS DONE IN 2D, BUT OUR ENTRY WAY HAS BEEN TO SAY, WELL, MOST CELLS IN THE BODY ARE IN 3D, AND WE'D LIKE TO ACTUALLY ENGINEER OR REGENERATE 3D TISSUES, WHAT'S THE EFFECT OF STIFFNESS ON CELLS IN 3D CONTEXT AND CAN WE USE WHAT WE LEARNED HERE TO IMPACT OUR STRATEGIES TO PROMOTE TISSUE REGENERATION? SO, THE MODEL SYSTEM THAT WE'RE USING IN THIS SYSTEM I'LL SPEND A COUPLE MINUTES EXPLAINING TO YOU. IT'S QUITE IMPORTANT FOR STUDIES WE PERFORM. AND THE MATERIAL IS SOMETHING IS ALGINATE, POLYSACCHARIDE DERIVED FROM SUGAR. YOU'LL SEEING A HYDRA GEL, AFM IMAGING POLYMER STRANDS, SPACES BETWEEN NANOPOROUS GELS, NANOMETER SPACING BETWEEN POLYMER STRANDS, WHAT HOLDS THE GEL TOGETHER IS POLYMER STRANDS CAN BIND CATIONS LIKE CALCIUM. ONLY THE G BLOCKS WITH BIND HE'S DIVALENT CATIONS AND DON'T CONTRIBUTE, THIS WILL BE IMPORTANT IN THE NEXT SLIDE. WE CAN CONTROL THE STIFFNESS AS INDICATED HERE BY ELASTIC MODULUS, BY CONTROLLING THE EXTENT OF CROSS-LINKING OF GEL , THE CONCENTRATION OF THE POLYMERS. THE REASON WE USE THIS SYSTEM IS BECAUSE IT ACTUALLY HAS A QUITE INTERESTING AND PROBABLY UNIQUE PRODUCT AMONG BIOMATERIALS USED IN THIS SPACE. I'LL TAKE A STEP BACK TO EXPLAIN. HOW PEOPLE HAVE BEEN MEHANOTRANSDUCTION STUDIES HISTORICALLY, TAKEN MOLECULES LIKE COLLAGEN HIGHER CONCENTRATION TO ALTER STIFFNESS. THEY CAN INCREASE STIFFNESS AS THEY INCREASE AMOUNT OF COLLAGEN. IF YOU THINK ABOUT IT, SINCE THE CELLS CAN ATTACH TO COLLAGEN, YOU'RE ALSO INCREASING THE DENSITY OF THE LIGANDS THAT CELLS CAN ATTACH TO AS YOU INCREASE AMOUNT OF COLLAGEN AND SOMETHING CALLED THE MESH SIZE, THE SPACING BETWEEN THE POLYMER CHAINS, ALSO DECREASED, AND THIS CAN AFFECT A FUSIONAL TRANSPORT OF THINGS LIKE GROWTH FACTORS. SO, WHEN YOU DO MECHANOTRANSDUCTION STUDIES IN COLLAGEN GEL SYSTEM, YOU DON'T KNOW WHICH IS CONTROLLING THE NET BEHAVIOR OF THE CELL. ALTERNATIVE APPROACH IS TO USE SYNTHETIC POLYMER, COUPLE CELL ADHESION LIKE AND, FOR EXAMPLE SMALL PEPTIDES WITH RGD SEQUENCE, KEEP LIGAND DENSITY CONSTANT, THAT ALLOWS US TO REGULATE THE STIFFNESS. AGAIN NOW WE END UP CHANGING THIS MESH SIZE SO WE CHANGE TRANSPORT OF THINGS LIKE GROWTH FACTORS. IT'S INTERESTING ABOUT THE ALGINATE IT ALLOWS US TO DECOUPLE. WHEN YOU HAVE CROSS-LINKING, IT CROSS-LINKS IN THE G BLOCKS. IF YOU ADD A LITTLE BIT OF DIVALENT CAT-IONS, YOU GET RED DOTS, A FEW CROSS-LINKS, THE WHOLE STRUCTURE LINED UP. NOW AS YOU ADD MORE DIVALENT CATIONS, YOU GET MORE CROSSLINKS, A STIFFER MATERIAL, BUT YOU DON'T CHANGE THE MESH SIZE AND IF YOU COUPLE SMALL ADHESION LIKE AND FOR EXAMPLE RGD-CONTAINING PEPTIDES LIGAND DENSITY WILL STAY THE SAME. THIS IS A WONDERFUL MODEL SYSTEM THAT ALLOWS US TO DECOUPLE MECHANICS FROM CHEMISTRY AND ARCHITECTURE IN THE GELS. HOW WE MEDIATE CELL ADHESION IN A PRECISE MANNER IS WE TAKE THE POLYMER CHAINS THAT OTHERWISE DON'T ALLOW FOR CELL ADHESION AND COVALENTLY CONJUGATE SMALL PEPTIDES THAT CONTAIN BINDING MOTIF OF INTEREST TO BIND TO RECEPTORS WE'RE INTERESTED IN EXPLORING. THIS TAKES GELS THAT OTHERWISE NON-ADHESIVE TO CELLS AND MAKES THEM HIGHLY ADHESIVE TO CELLS. THAT'S THE SYSTEM. NOW, THE FIRST PIECE OF DATA WE GENERATED THAT INDICATED THAT ACTUALLY THIS IDEA OF CONTROLLING STIFFNESS WAS A POWERFUL APPROACH TO CONTROL BIOLOGY OF CELLS CAME FROM WORK OF NATE'S, NOW RUNNING HIS OWN LAB AT WASH U, PUBLISHED NINE YEARS AGO. THE EXPERIMENT WAS SIMPLE. TOOK MESENCHYMAL STEM CELLS, PUT THEM IN A SOFT GEL, THEY BECAME ADIPOCYTES, FAT CELLS. IF HE PUT THEM IN GELS AT INTERMEDIATE MODULUS, THEY BECAME OSTEOBLASTS. THIS WAS DEPENDENT ON THE ADHESION LIGAND DENSITY SO THE CELLS HAVE TO BE ABLE TO ATTACH AND PULL ON THE MATERIAL TO FEEL THE STIFFNESS AND THE MORE THEY CAN ATTACH, THE MORE THEY CAN PULL, THE MORE SENSITIVE THEY WERE TO MECHANICAL PROPERTIES. THIS IS JUST SOME GENE EXPRESSION CHARACTERIZATION TO CONFIRM THIS STAINING INDICATION. SO, SHOWING MECHANICS MATTERS, NOT JUST IN VITRO. IN A FOLLOW-UP PAPER TRANSPLANTED VERY SMALL NUMBERS OF MESS OF MESENCHYMAL STEM CELLS IN HYDRA GELS INTO CRANIAL DEFECTS, THE GELS HAD A STIFFNESS THAT WE THOUGHT WOULD LEAD TO OSTEOGENESIS, ONE THAT WOULD BE OPTIMAL FOR -- EXCUSE ME, ADIPOGENESIS OR BE TOO STIFF. HE COULD GET MORE BONE FORMING WITH THE CELLS FOR OSTEOGENESIS, WE CAN CONTROL STEM CELL BEHAVIOR IN CULTURE, ALSO CONTROL HOW STEM CELLS CONTRIBUTE TO REGENERATION IN IN VIVO CONTEXT BY CONTROLLING THE MECHANICAL PROPERTIES. NOW, THESE WERE INTERESTED FINDINGS, PARTICULARLY AT THE TIME. BUT THEY IGNORE KEY ASPECTS OF THE BIOLOGY. IN PARTICULAR WHAT THESE IGNORE IS THE FACT THAT THE ECM IN ITS PHYSICAL PROPERTIES ARE QUITE A BIT MORE COMPLEX THAT WHAT PEOPLE HAD BEEN THINKING OF. WE TALK ABOUT STIFFNESS HAVING A BIG IMPACT ON CELL BIOLOGY, WHAT WE'RE REALLY DOING IS ASSUMING TISSUES ARE PURELY ELASTIC. WE'RE ASSUMING THEY ACT LIKE A RUBBER BAND. IF WE STRETCH IT, WE HAVE TO KEEP APPLYING A FORCE TO MAINTAIN ITS STRETCH AND AS SOON AS WE LET GO IT GOES BACK TO ITS ORIGINAL SIZE AND SHAPE, BUT THAT RUBBER BAND STORES THE ENERGY WE PUT INTO IT AND WILL ELASTICALLY GO BACK. THAT'S WHAT YOU SEE IN THE HYDROGELS. IF YOU LOOK AT FAT TISSUE, LIVER, BRAIN, REPRESENTATIVE OF THE MICROENVIRONMENT WHICH BONE REGENERATION HAPPENS, THEY ARE NOT PURELY ELASTIC. INSTEAD THEY ARE WHAT WE CALL VISCO ELASTIC, BETWEEN A LIQUID AND SOLID. HOW WE CAN DEMONSTRATE, THE STRESS RELAXATION TEST, WE APPLY A GIVEN DEFORMATION, STRETCH THAT RUBBER BAND, AND THEN WE MEASURE HOW MUCH FORCE WE HAVE TO APPLY TO KEEP IT STRETCHED. WHAT YOU CAN APPRECIATE IS THESE TISSUES AND THIS COLLAGEN GEL, THEY REMODEL OVER TIME, DISSIPATE THE STRESSES, AND SO AT THE END YOU DON'T HAVE TO APPLY ANY STRESS TO HAVE IT STAY IN DEFORMED STATE. THAT LED US TO BEGIN TO ASK THE QUESTION, DO CELLS EVEN CARE? IS IT ONLY THE STIFFNESS THAT MATTERS OR ARE THESE TIME DEPENDENT PROPERTIES IMPORTANT TO CELLS AND TO TISSUES? TO GET CONTROL OVER THE RATE, I DON'T GO INTO GREAT DETAIL BUT WE MODULATE THAT PROPERTY IN GELS BY CONTROLLING THE MOLECULAR WEIGHT OF THE POLYMER CHAINS AND THAT COUPLING SMALL PEG MOLECULES THAT INTERFERE WITH THE WAY THE CHAINS SLIDE WITH EACH OTHER. I WON'T GO DETAILS BUT WE CAN CREATE FAMILIES OF HYDROGELS WITH THE SAME INITIAL ELASTIC MODULUS OR STIFFNESS BUT RELAX OVER DIFFERENT TIME SCALES, HALFTIME FOR RELAXATION. YOU SEE THEY VARY IN THIS PARTICULAR SET FROM 3300 SECONDS DOWN TO ABOUT 70 SECONDS, OR A MINUTE. SO WE MADE THESE GELS, PUT CELLS WITHIN THEM, AND WE STUDIED TO SEE IF THE CELLS' BEHAVIOR CHANGED. YOU SEE RESULTS OF JUST LOOKING AT MORPHOLOGY OF CELLS THAT WE'VE PLACED INTO THE GELS, WHERE WE HAVE FASTER RELAXING GELS TO THE RIGHT, OTHERWISE THESE CELLS ARE EXACTLY THE SAME ACROSS EACH PANEL IN TERMS OF LIGAND DENSITY AND INITIAL STIFFNESS. WHAT CAN YOU PRESSURE, CELLS IN THE SLOWLY RELAXING GELS STAY ROUNDED UP BALLS, DON'T EXTEND, SPREAD OR MOVE AROUND IN THE GELS. THIS IS AN OBSERVATION THAT'S MADE OVER AND OVER AND OVER AGAIN WHEN PEOPLE PUT CELLS INSIDE HYDRAGELS, THEY LOOK LIKE THIS. WHAT'S STRIKING, YOU GO TO VERY RAPIDLY RELAXING GELS, CELLS BEGIN TO SPREAD, YOU DON'T SEE IT HERE BUT THEY MIGRATE AND PROLIFERATE QUITE ACTIVELY. A KEY FEATURE OF THE GELS THAT I SHOULD MENTION TO YOU IS CELLS CAN'T PROTEOLYTICALLY DEGRADE THESE GELS. WE'RE NOT SEEING THIS ACTIVITY BECAUSE CELLS ARE DEGRADING THE GEL, WE'RE SEEING IT BECAUSE THE CELLS ARE PHYSICALLY REMODELING AND PUSHING THE GEL AROUND AND MOVING IT TO ENABLE THEMSELVES TO SPREAD, MIGRATE, PROLIFERATE WITHIN THE GEL. NOW, THIS BEHAVIOR IS DEPENDENT ON THE CELL'S ABILITY TO ADHERE, GRAB HOLD OF THE MATERIAL, IF THERE'S NO CELL ADHESION SITES ON THE POLYMER CHAINS THE CELLS STAY VERY ROUNDED UP, EVEN IN THE RAPIDLY RELAXING GELS. SO FUNDAMENTAL CELL BEHAVIOR IS DRAMATICALLY ALTERED BY VISCOELASTICITY. HERE WE TAKE MESENCHYMAL STEM CELLS, PUT THEM IN A SERIES OF CELLS WITH RAPID RELAXATION, TO THE RIGHT, TWO INITIAL STIFFNESSES. YOU CAN SEE STIFFNESS DOES STILL MATTER. IF THE GELS ARE TOO STIFF NONE OF THE CELLS BECOME ADIPOCYTES, HOW MANY IS DEPEND ON RATE OF RELAXATION, AS YOU CAN SEE VISUALLY, QUANTITATIVELY DOWN HERE. IN THIS CASE, ACTUALLY ADIPOGENESIS IS INHIBITED WITH RAPID RELAXATION. OSTEOGENESIS IS ENHANCED AS THE MATERIALS EXHIBIT A MORE RAPID RELAXATION BEHAVIOR AS INDICATED WITH THE QUANTIFICATION OF ALP EXPRESSION, HERE YOU SEE VISUALLY. FOR THOSE IN THE BONE AREA, IF YOU LOOK AT MINERALIZATION, COLLAGEN DEPOSITION, AND COMPOSITION, WE FIND IF WE GO TO -- PUT CELLS INSIDE GELS THAT CONTRIBUTE RAPID RELAXATION, WE GET INTERCONNECTED MINERALIZE AND COLLAGEN RICH MATRIX IN VITRO, NOT IN THE SAME GELS THAT JUST RELAX MORE SLOWLY. WE'RE SEEING FUNDAMENTAL CHANGES IN CELL BEHAVIOR, SIMPLY BY ALTERING RATE OF RELAXATION. IS THERE A PHYSIOLOGIC RELEVANCE SORE THIS ARTIFACT OF THESE SYSTEMS THAT WE'RE CREATING IN THE LABORATORY? TO BEGIN TO ADDRESS THIS, WE WENT BACK AND BEGAN TO CHARACTERIZE THE MICROENVIRONMENTS IN WHICH BONE REGENERATION NORMALLY HAPPENS IN THE BODY. SO FOR EXAMPLE WE TOOK COAGULATED MARROW FROM RATS, OBTAINED FRACTURED HEMATOMAS FROM PATIENTS COMING INTO THE HOSPITAL IN BERLIN FOR REVISION SURGERIES, AND WE SUBJECTED THEM TO THE SAME TYPES OF STRESS RELAXATION RELAXATION TEST SHOWN FOR THE GELS. THIS IS WORK BY MAX AND EVEY IN BERLIN. THE MATERIALS HAVE EXACTLY THE SAME TYPE OF STRESS RELAXATION BEHAVIOR AS HYDRAGELS AND RATE OF RELAXATION MATCHES WITH WHAT IS OPTIMAL FOR OSTEOGENESIS. WHAT WE'RE PROBABLY DOING WITH OUR SYSTEMS IS SIMPLY REDISCOVERING THE KIND OF MICROENVIRONMENT THAT OUR BODY NORMALLY USES TO TRY TO ENHANCE OSTEOGENESIS AND BONE REGENERATION IN AT LEAST THESE CONTEXTS. NOW, TO FURTHER CONFIRM THIS ACTUALLY HAD SOME RELEVANCE IN PHYSIOLOGIC SENSE, WE TOOK TWO TYPES OF GELS WITH THE SAME STIFFNESS, ONE THAT EXHIBITED SLOW RELAXATION, THE OTHER EXHIBITED FAST RELAXATION, AND AGAIN TRANSPLANTED SMALL NUMBERS OF MSCs IN THEM AND FOUND MUCH MORE BONE FORMATION IN VIVO WITH FAST RELAXING GEL AS VERSUS THE SLOWLY RELAXING GEL, INDICATING NOT JUST IN VITRO BUT ALSO IN VIVO WE CAN ALTER BEHAVIOR AND REGENERATION THAT ARISES FROM STEM CELLS, BY CONTROLLING THIS ASPECT OF MATERIALS TO TRANSPLANT THE CELLS. NOW, WE'VE BEEN INTERESTED IN TRYING TO UNDERSTAND MECHANISTICALLY HOW THESE WORK. I WON'T GO INTO DETAIL BUT WILL HIGHLIGHT A COUPLE THINGS WE KNOW AT THIS POINT. MUCH IS STILL NOT KNOWN. ONE OF THE FEATURES THAT WE PREDICT BASED ON MATHEMATICAL MODELING IS THAT WHEN CELLS REACHED OUT AND GRABBED AND THEN BEGAN TO PULL ON THESE MATRICES IF THEY EXHIBIT RAPID RELAXATION WILL CLUSTER, CREATING A MORE RICH ENVIRONMENT FOR ADHESION DIRECTLY AROUND THEMSELVES. WE STUDIED THIS USING FRET TECHNIQUES, WHAT YOU'RE LOOKING AT IN THE TOP PANEL HERE IS CELLS IN A GEL THAT EXHIBIT SLOW RELAXATION, HERE IS THE NUCLEUS, HERE IS THE SURROUNDING GEL, IT ALL LOOKS THE SAME. IN CONTRAST, IF THE SAME CELLS PUT INTO A GEL EXHIBIT RAPID RELAXATION, YOU CAN APPRECIATE THE RED HALO INDICATING THE CELL IS REMODELING AS PREDICTED, INCREASING IN THIS CONTEXT AND THIS IS A QUANTIFICATION OF THE FRET SIGNAL. PART OF THE MECHANISM IS CELLS ARE PHYSICALLY REMODELING THE MATERIAL AND CHANGING MICROENVIRONMENT. FROM AN ENGINEERING PERSPECTIVE, ONE OF THE THINGS THAT MIGHT OCCUR TO YOU, IF THE CELLS ARE DOING THIS AND REARRANGING THE MATERIAL, MAYBE ALL WE'RE DOING IS ACTUALLY RECREATING A STIFF MATERIAL AROUND THE CELL, MAYBE THIS IS STIFFNESS STILL AND NOT VISCALELASTICITY. WE HAVE TO BE ABLE TO MEASURE MECHANICAL PROPERTIES, VERY SMALL REGION, MICRONS AROUND A CELL WITHIN A 3D MATERIAL. AND SO WE DIDN'T THINK WE WERE GOOD ENOUGH BIOLOGISTS OR ENGINEERS TO DO THAT SO WE DECIDED TO TAKE A DIFFERENT APPROACH TO ADDRESS THIS QUESTION, WHETHER REALLY THIS IS JUST CHANGING IN STIFFNESS AT THE END OF THE DAY, AND THE WAY WE APPROACH THIS WAS JUST TO DO GLOBAL GENE EXPRESSION ANALYSIS USING RNA SEQUENCING. AND WE CREATED A GRID WHERE WE VARIED RATE OF STRESS RELAXATION, CHANGED THE STIFFNESS OF THE GELS, CHANGED THE DENSITY OF THE ADHESION LIGANDS, DID RNA-SEQ, LOOKED TO SEE DIFFERENCES IN GENE EXPRESSION. JUST FOCUSED ON THIS DATA HERE, WHICH IS THE SET OF DIFFERENTIALLY EXPRESSED GENES WITH EACH VARIABLE. IF STRESS RELAXATION JUST ALLOWED CELLS TO REMODEL THE GEL, AND CREATE A HIGH DENSITY OF ADHESION LIGAND, STIFFER SUBSTRATE AROUND THE CELLS, THE GENE EXPRESSION CHANGE WITH RELAXATION SHOULD OVERLAP COMPLETELY WITH CHANGES IN STIFFNESS AND CHANGES IN ADHESION LIGAND DENSITY. NOW, WE DO SEE THERE IS SOME OVERLAP BETWEEN THE IMPACT OF STRESS RELAXATION AND THAT OF CHANGINGED A LESION LIGAND DENSITY, THAT MAKES SENSE, CELLS DO INDEED GATHER LIGANDS AROUND THEMSELVES, BUT OVER THE CONDITIONS THAT WE DID THESE EXPERIMENTS WE SEE PRETTY MUCH NO OVERLAP BETWEEN GENES DIFFERENTIALLY REGULATED BY STIFFNESS AND THOSE DIFFERENTIALLY REGULATED BY VISCOELASTICITY, SUGGESTING DIFFERENT MECHANISMS AT PLAY WITH VISCOELASTICITY VERSUS THOSE OF STIFFNESS. NOW, THIS BEHAVIOR WE'RE LOOKING AT I'VE SHOWN YOU DATA WITH MSCs, THIS SEEMS TO BE A BROADLY RELEVANT BEHAVIOR. IN THIS CASE THEY RESPOND TO NEURAL RELAXATION, NOT STIFFNESS AND LIGAND DENSITY, OTHER CELL TYPES THE DEPENDENCY IS DIFFERENT FOR EACH CELL TYPE BUT WE SEE DEPENDENCY ON ALL THESE FACETS. RECENTLY WE BEGAN TO EXPLORE OUTSIDE STEM CELL RANGE. KYLE VINING, DOING HIS Ph.D. RIGHT NOW AT HARVARD, LOOKING AT MONOCYTE AND DIFFERENTIATION INTO DENDRITIC CELLS, PUTTING THEM IN CELLS, VARYING STIFFNESS ANDS EXTENT OF RELAXATION. WHAT HE'S BEEN ABLE TO SHOW, IF WE PUT CELLS INSIDE GELS HIGHLY ELASTIC, THESE NTBZ GELS, HE GETS SIGNIFICANT UPREGULATION OF MARKERS OF DIFFERENTIATION OF MONOCYTES IN PATHWAY TOWARDS DENDRITIC CELLS, IN CONTRAST PUTS THEM IN CELLS WITH THE SAME STIFFNESS BUT ARE VISCOELASTIC YOU GET GET THE SAME LEVEL OF RESPONSIVENESS. MONOCYTES THAT DIFFERENTIATE AND BECOME ANTIGEN PRESENTING CELLS IS DRAMATICALLY IMPACTED NOT JUST BY STIFFNESS BUT WHETHER OR NOT THE MATERIAL IS VISCOELASTIC OR PURELY ELASTIC. LOOK AT GLOBAL PATTERN OF GENE EXPRESSION, IN VISCOELASTIC SUBSTRATE MARKERS INDICATE IMMATURE DENDRITIC CELLS, MATURATION MARKERS IN THE STIFFER SUBSTRATES, WE THINK PROBABLY RELEVANT IN AMINO ACID THERAPY, CANCER, HALLMARKS OF TUMORS THEY BECOME STIFFER, THEY BECOME MORE ELASTIC AS THEY DEVELOP. NOW, ANOTHER MECHANISM OR ASPECT OF THIS, EVERYTHING I'VE BEEN DESCRIBING HAS BEEN CELL'S ABILITY TO REACH OUT, GRAB, AND PULL. WHO STARTED THIS WORK IN THE LAB, HIS PICTURE EARLIER, NOW A PROFESSOR AT STANFORD WITH A PROFESSOR AT JOHNS HOPKINS NOW DID THIS WORK IN THE LAB, CONTINUING TO DO THIS WORK IN COLLABORATION WITH US, IN HIS OWN LAB IN CARTILAGE FORMATION. AN INTERESTING FEATURE, HE'S PUTTING CELLS INSIDE GELS THAT DON'T HAVE ADHESION LIGANDS, ALL THEY CAN DO IS PUSH ON THE GEL, NOT PULL. HE FOUND WAS, AGAIN, ABILITY OF THESE CELLS TO ACTUALLY FORM CARTILAGINOUS TISSUES IN VITRO WAS IMPACTED BY THE RATE OF STRESS RELAXATION AND YOU HAD MUCH BETTER TISSUE FORMATION IN STRESS RELAXING GELS THAN IN MORE ELASTIC SUBSTRATES TYPICALLY USED FOR 3D CULTURE. WHAT HE SHOWED WAS THAT THIS IS DUE TO THE FACT WHEN CELLS ARE IN ELASTIC SUBSTRATE AND TRY TO LAY DOWN MATRIX OR PROLIFERATE, AS I MENTIONED EARLIER, THESE ACT LIKE RUBBER BANDS. SO THE CELL PUSHES, AND THE MATERIAL PUSHES BACK. THIS ENDS UP LEADING TO EXPRESSION OF IL-1 BETA, AND THROUGH A SERIES OF EVENTS ENDING UP INCREASING THINGS LIKE CELL DEATH, EXPRESSION, CATABOLIC STATE OF THE CELLS. IN CONTRAST IN VISCOELASTIC MATERIAL THEY PUSH AGAINST SUBSTRATE, IT REMODELS, DISSIPATES ENERGY, DOESN'T KEEP PUSHING BACK AT THEM. THIS ALLOWS THE CELLS TO ACTUALLY CONTINUE TO GROW, PROLIFERATE, MAKE MATRIX AND TURN INTO A TISSUE. OVERALL HOPEFULLY A SENSE OF HOW THE MECHANICAL PROPERTIES IN PARTICULAR VISCOELASTICITY IS A KEY VARIABLE IN MANY EARS OF BIOLOGY, NATURALLY AS WELL AS IN THE DESIGN OF BIOMATERIALS, WE MIGHT WANT TO CREATE, TO PROMOTE TISSUE REGENERATION. THAT'S THE FIRST PART OF WHAT I WANTED TO TALK ABOUT IN TERMS OF MECHANICS. I WANT TO SPEND A FEW MINUTES TALKING ABOUT SOMETHING DIFFERENT BUT RELATED. THAT IS WHETHER WE CAN ACTUALLY APPLY EXTERNAL FORCES ONTO CELLS AND TISSUES TO CONTROL REGENERATIVE BEHAVIOR. UNTIL THIS POINT WE'VE BEEN TALKING ABOUT WHEN A CELL PULLS HOW MUCH RESISTANCE IT FEELS. NOW WE'LL TALK ABOUT ACTIVELY PUSHING ON CELLS. THIS WORK GOT STARTED THROUGH THE Ph.D. THESIS RESEARCH OF ELLEN ROACH, A TALENTED STUDENT, NOW RUNNING A LAB AT M.I.T. THIS WORK WAS ALL IN COLLABORATION WITH WALSH, ROBOTICS FACULTY AT HARVARD, SUPPORTING FUNCTION OF FAILING HEARTS, MORE AS A MECHANICAL SUPPORT SYSTEM BUT WE BEGAN TO THINK ABOUT WHETHER THESE MECHANICAL SYSTEMS MIGHT BE USEFUL NOT JUST TO PROVIDE SHORT-TERM MECHANICAL SUPPORT BUT TO ACTUALLY INDUCE ACTIVE REGENERATION. WE'VE BEEN EXPLORING THIS, LARGE DEFECTS OF SKELETAL MUSCLE TISSUE UNFORTUNATELY ARE TOO FREQUENT AND REQUIRE THERAPY, AND THERE'S A VARIETY OF STRATEGIES WE CURRENTLY UTILIZE TO TRY TO PROMOTE REGENERATION OF SKELETAL MUSCLE. THEY ALL HAVE LIMITATIONS. EXPLORING THIS IDEA OF CONTROLS MECHANICAL STIMULATION USING SOFT ROBOTICS TO APPLY KNOWN AND ESTABLISHED AND REPRODUCIBLE FORCES AND STRAINS TO TISSUES, IN ORDER TO STUDY HOW THEY CAN PERHAPS INDUCE REGENERATION. THE KIND OF SYSTEM SHOWN HERE SCHEMATICALLY, HERE A RODENT, ACTUATOR SYSTEM, STRESS AND STRAIN OVER TIME, IN THIS CASE MORE OUTSIDE THE BODY, OR INSIDE THE BODY. OUR PARTICULAR INTEREST HERE THIS COULD PROVIDE MINIMALLY INVASIVEMEANS TO PROMOTE REGENERATION BY APPLYING A SPECIFIC FORCE ON THE EXTERIOR OF THE ANIMAL, ULTIMATELY PERHAPS THE PERSON. I'LL SKIP THE BACKGROUND BUT BORI AND CHRISTINE WHO STARTED THIS IN THE LAB WERE ABLE TO EXPLORE A WIDE RANGE OF PARAMETER SPACE IN TERMS OF TYPES OF -- MAGNITUDE OF FORCES, FREQUENCY, DURATION, ABLE TO FIND SPECIFIC CONDITIONS THAT WERE INDEED ABLE TO ACTIVELY PROMOTE REGENERATION, LOOKING AT HISTOLOGY OF SKELETAL MUSCLE TISSUE THAT DOES NOT HAVE MECHANICAL STIMULATION OR IS MECHANICALLY STIMULATED AND WHAT YOU CAN APPRECIATE IS WITH MECHANICAL STIMULATION IF WE LOOK OVER TIME WE SEE UPREGULATION OF THE NUMBER OF PAX 7 CELLS, SATELLITE OR STEM CELLS IN MUSCLE, WE'RE GETTING INCREASING NUMBERS OF STEM CELLS, AND IF WE LOOK AT CENTRALLY LOCATED NUCLEI WHICH IS INDICATION OF ACTIVE REGENERATIVE PROCESS IN MUSCLE YOU SEE ACTIVE INCREASES WITH MECHANICAL STIMULATION. OVER TIME WHAT WE SEE IS SIGNIFICANT DIMINISHMENT IN CALCIFICATION IN THE MUSCLE AND FIBROSIS WHEN WE APPLY MECHANICAL STIMULATION MS VERSUS CONTROL, WITH SIGNIFICANT IMPACT IN SEVERE COMBINED, IN THIS CASE, MYOTOXIN IN ISCHEMIC INJURY TO THE SKELETAL MUSCLE. THE NET RESULT IS WE GET AN IMPROVED FUNCTIONALITY, LOOKING AT THE FORCE THE MUSCLE IS CAPABLE OF GENERATING, FOLLOWING INJURY, WE LOSE ALMOST COMPLETE FUNCTION IN THIS MODEL, YOU GET SOME SPONTANEOUS RECOVERY OVER TIME IN THE GRAY, SIGNIFICANT ACCELERATION OF FUNCTIONAL RETURN OF FUNCTION, INDICATED BY MUSCLE -- FORCE GENERATION WITH MECHANICAL STIMULATION HERE IN RED. SO WE'RE ABLE TO GET SIGNIFICANT IMPACT IN THE CONTEXT OF YOUNG ANIMALS, AGED ANIMALS WE'VE DONE THIS NOW, IN MODELS OF MUSCULAR DYSTROPHY, SO IT TEAMS TO BE A BROADLY APPLICABLE CONCEPT IN MUSCLE REGENERATION, WE'RE BEGINNING TO EXPLORE THIS NOW IN BONE REGENERATION AS WELL. BUT THE MYSTERY IS HOW DOES THIS WORK, WHAT'S ACTUALLY HAPPENING. THERE'S MANY POSSIBLE MECHANISMS HERE. AND PROBABLY MANY MECHANISMS ACTUALLY ARE RELEVANT. I'LL TELL YOU ABOUT ONE PATH THAT WE'VE BEEN RECENTLY EXPLORING, AND THAT IS TO EXPLORE WHETHER OR NOT THIS MECHANICAL STIMULATION MAY ALTER THE INFLAMMATORY RESPONSE THAT ONE GETS FOLLOWING INJURY. NOW, AS YOU KNOW FOLLOWING INJURY, YOU HAVE NEUTROPHILS COMING IN AS EARLY RESPONDERS, EXTORT TO GET MONOCYTES, MACROPHAGES, ADAPTIVE CELLS COMING IN OVER TIME DURING THESE PROCESSES OF INFLAMMATION AND ULTIMATELY REGENERATION. THEY HAVE BEEN ASKING WHETHER IMMUNE RESPONSES MEDIATE SOME ASPECT OF THE MECHANICAL RESPONSE THAT WE'RE SEEING IN TERMS OF REGENERATION. ONE OF THE FIRST THINGS SHE DID WAS TO DO A REALLY SIMPLE EXPERIMENT WHERE SHE JUST ANALYZED ALL THE CYTOKINES, SIGNALING MOLECULES IN THE DAMAGED MUSCLE TISSUE AND COMPARED MECHANICAL STIMULATION VERSUS CONTROL. AND WHAT YOU'RE LOOKING AT HERE IS BLUE MEANS IT'S DOWNREGULATED, RED IS UPREGULATED, THIS IS ALWAYS WITH MECHANICAL STIMULATION, RELATIVE TO CONTROL, AND SO WHAT YOU CAN APPRECIATE IS WHAT'S MECHANICAL STIMULATION, YOU GET DOWNREGULATION, VIRTUALLY ALL THE CYTOKINES SHE'S EXAMINED IN THE TISSUE IN THE SHORT TERM AFTER THE INITIATION OF THE MECHANICAL STIMULATION, OVER TIME YOU SEE SOME DOWNREGULATION BUT IT BEGINS TO NORMALIZE. IF YOU START TO LOOK AT WHAT KIND OF PATHWAYS THESE CYTOKINES ARE INVOLVED IN, THEY ARE INVOLVED IN A NUMBER OF SIGNALING PATHWAYS, LYMPHOCYTE CHEMOTAXIS, NEUTROPHIL CHEMOTAXIS. WHEN SHE LOOKED AT NEUTROPHILS FOUND A RAPID CHANGE IN THE NUMBER OF NEUTROPHILS AND SIGNIFICANT DIMINISHMENT WITH MECHANICAL STIMULATION, YOU CAN SEE THE TIME COURSE. EARLY AFTER THE INJURY WE HAVE NO IMPACT BUT YOU ACTUALLY CLEAR THE TISSUE OF NEUTROPHILS MORE RAPIDLY WITH MECHANICAL STIMULATION, AND IF YOU LOOK AT THE ACTIVATED NEUTROPHILS YOU SEE A SIGNIFICANT DIFFERENCE BETWEEN THESE. IT APPEARS WITH THIS MECHANICAL STIMULATION AT LEAST ONE OF THE MECHANISMS MAY BE THAT WE'RE CHANGING LOCAL CYTOKINE PROFILE PROBABLY BY PUMPING ACTION, CHANGING ACTIVITY OF LOCAL IMMUNE CELLS, THE QUESTION IS COULD THIS TIE TO REGENERATION. ONE SIMPLE WAY SHE'S EXPLORING IS TO LOOK IN VITRO AT THE INTERSECTION OF NEUTROPHIL BIOLOGY AND MUSCLE PROGENITORS BECAUSE LITTLE WAS KNOWN ABOUT WHETHER THE TWO SYSTEMS INTERACTED. SHE'S TAKING CONDITIONED MEDIA FROM NEUTROPHIL CULTURES, ACTIVATED NEUTROPHIL CULTURES, EXPOSING TO MUSCLE PROGENITOR CELLS IN CULTURE. YOU CAN APPRECIATE IF YOU LOOK AT THE EFFECT ON THE GROWTH OF THE CELLS, AS INDICATED BY PERCENT EDU POSITIVE, WITH THE CONDITIONED MEDIA, YOU GET A SIGNIFICANT INCREASE IN PROLIFERATION, AND GET A SIGNIFICANT DECREASE IN MARKERS OF DIFFERENTIATION. IN THIS CASE LOOKING AT THE CELLS IN CULTURE, AND SO THE WORKING PREMISE RIGHT NOW IS NEUTROPHILS ENHANCE PROLIFERATION OF SATELLITE CELLS, PREVENT DIFFERENTIATION, BY CLEARING CELLS MORE RADICALLY ALLOWING TISSUES TO MOVE INTO LATER STAGES OF REGENERATION MORE RAPIDLY AND INDUCE RETURN OF FUNCTION IN A RAPID MANNER. WE'RE TRYING TO EXPLOIT MECHANOREGENERATION. WE CAN TAKE STEM CELLS, SYNTHETIC NICHES, CONTROL STIFFNESS AND USE THESE AS MEANS TO TRANSPLANT CELLS TO TRY TO PROMOTE REGENERATION. THERE'S A LOT OF WORK IN MANY LABORATORIES WHERE THEY ARE TAKING CELLS OF INTEREST, PRECONDITIONING THEM OUTSIDE THE BODY, ON MATERIALS THAT HAVE SPECIFIC TYPES OF PROPERTIES, SYSTEMIC DELIVERY. I'VE TALKED HOW WE'RE TRYING TO USE ACTIVE MECHANICAL SYSTEMS TO IMPOSE MECHANICAL LOADS ON THE TISSUES IN THE BODY. NOW, WITH ALL THIS WORK ONE OF THE THINGS THAT WE'VE STUMBLED INTO IS THAT WE'RE OFTENTIMES TAKING MATERIALS AND WE'RE ACTUALLY HAVING TO PUT THEM IN CONTACT WITH TISSUES INSIDE THE BODY, OR ON THE BODY, AND AS MANY OF YOU ARE AWARE ADHESION ON WET AND DYNAMIC MATERIALS SUCH AS TISSUE SURFACES IS A BIG CHALLENGE IN THE MEDICAL FIELD. JANE LEE WHO CAME TO THE LAB THREE YEARS AGO BECAME INTERESTED IN COULD WE DEVELOP BETTER TIMES OF ADHESIVE MATERIALS, AND WHEN HE STARTED LOOKING AROUND FOR INSPIRATION, FOR HOW ONE MIGHT BE ABLE TO SOLVE THIS PROBLEM, HE FOUND THIS PARTICULAR ORGANISM, THE SLUG, SECRETES A MUCUS TO MOVE AROUND, IT WILL CHANGE THE COMPETITION WITH A PREDATOR AND ADHERE STRONGLY TO SUBSTRATE SO PREDATOR CAN'T PULL IT OFF AND EAT IT. THIS WAS CHARACTERIZED AS HYBRID NETWORK, POLYSACCHARIDES AND PROTEINS. THIS WAS VERY SIMILAR TO A POLYMER SYSTEM THAT WE'VE BEEN WORKING ON ALREADY, AND THAT WAS INSPIRED BY SOME OF THE VISCOELASTIC STUDIES WE'VE BEEN DOING FOR SEVERAL YEARS. AND TO EXPLAIN THIS I NEED TO GO BACK A LITTLE BIT AND ALL THESE HYDRA GELS I'VE BEEN DESCRIBING TO THIS POINT IN TIME FOR TRANSPLANTING CELLS AND MANIPULATING CELLS THEY ARE MAINLY WATER, 98% WATER, 2% POLYMERS TYPICALLY, NOT SURPRISINGLY THEY ARE REALLY WEAK. HYDRA GELS ARE KNOWN TO BE A WEAK CLASS OF MATERIALS. AND WE WANTED TO EXPLORE WAYS OF TRYING TO ENHANCE PROPERTIES AND HE WAS IN THE LAB AT THE SAME TIME NATE WAS DURING THE FIRST WORK ON VISCOELASTICITY AND STIFFNESS. HE SAID THE PROBLEM FROM MECHANICS PERSPECTIVES, MEDICAL ADHESIVE, IF WE HAVE MEDICAL ADHESIVE AND, FOR EXAMPLE, SOMETHING THAT MIGHT FORM, YOU HAVE A CRACK, ALL THE ENERGY OF THE CRACK IS IN A SMALL REGION, CONCENTRATED, YOU HAVE HIGH STRESSES, THAT LEADS TO PROPAGATION OF THE CRACK THROUGH THE MATERIAL, FAILS IN BRITTLE MANNER, WEAK AND JUST NOT TOUGH. HE RECOGNIZED WHAT'S INTERESTING ABOUT ALGAEINIC GELS CROSS-LINKED IN VISCOELASTIC, DECROSS LINKING WOULD ABSORB ENERGY. HE SAID THIS IS INTERESTING FROM A MECHANICS PERSPECTIVE, A WAY OF ABSORBING ENERGY. IF WE COMBINE THESE TYPES OF ELASTIC SUBSTRATES THAT THEY CAN TRANSFER STRESSES TO THESE IONICALLY CROSS-LINKED NETWORKS THAT DISSIPATE SUPPRESS CAN POTENTIALLY DISSIPATE IN A LARGE VOLUME OF GEL AND MAKE THE GELS REALLY TOUGH. SO THIS IS INSPIRED BY BIOLOGY BUT ACTUALLY, YOU KNOW, WAS NOT INTENDED TO BE A BIOLOGICAL OUTCOME. NOW, HE ACTUALLY WENT AHEAD AND MADE SOME GELS, AND THIS IS JUST GIVING YOU A SENSE OF WHAT KINDS OF GELS HE COULD CREATE, 1 MILLIMETER PARTICULAR HYDRA GEL, AND ONE INCH STEEL BALL DROPPED FROM HERE, IT DOESN'T MAKE THE HYDRA GEL FAIL, IT SUPPORTS THE LOAD AND BOUNCES OVER A ERRED POOH OF TIME. IT WAS STRIKING WHEN HE REALIZED SLUGS HAD THE STUCKEY MUCUS, CROSS-LINKED NETWORK COMBINED WITH IONICALLY CROSS-LINK POLYSACCHARIDE SOUNDS LIKE THE TOUGH GELS THAT THEY HAD BEEN MAKING. WE COULD JUST USE THESE AND MAKE A NEW TYPE OF ADHESIVE SO WE HAVE THIS TOUGH GEL THAT COULD DISSIPATE LOTS OF ENERGY, BE REALLY TOUGH, AND THEN WE NEED THOUGH TO INTERFACE IT WITH A TISSUE, AND SO WHAT HE PROPOSED IS TO COMBINE SOME TYPE OF MOLECULES THAT WOULD BOND WELL BOTH TO THE TISSUE, TISSUE BELOW IT, TOUGH GEL ABOVE IT. MOLECULES WOULD A HIGH POSITIVE CHARGE WOULD BE ABLE TO INTERACT WITH TISSUE AND GEL, COVALENTLY CONJUGATE WITH BOTH TO FORM INTERFACE TO TRANSFER STRESS BETWEEN THE TISSUE AND GEL. WE WOULD HAVE GELS, PREFABRICATE THEM, COAT THEM WITH GREEN SOLUTION, HOPEFULLY IT BINDS AND FORMS A TOUGH MATERIAL. ONE WAY TO QUANTIFY IS INTERFACIAL TOUGHNESS, INCREASE IN THESE ADHESIVES AS FUNCTION OF TIME, IT TAKES A MINUTE OR TWO FOR IT TO ADHERE WELL, HERE IS COMPARISON TO SUPER GLUE THAT MANY OF YOU MAY BE FAMILIAR WITH, THAT'S THE STRONGEST ADHESIVE OUT THERE TODAY. YOU CAN SEE WITHIN A MINUTE OR SO WE START TO DRAMATICALLY OUTPERFORM THE PERFORMANCE OF THAT. IF WE COMPARE MORE BROADLY TO ALL ADHESIVE OUT THERE, IN TERMS OF ADHESION ENERGY, HERE IS WHAT WE'RE CALLING THIS TOUGH ADHESIVE, HE'S IS CYANO ACROLATE, COMMERCIALLY AVAILABLE FIBER INVASIVES AND NANOPARTICLES UNDER DEVELOPMENT. YOU CAN SEE WE DRAMATICALLY OUTPERFORM WHEN WE ADHERES IN THIS CASE, ALL THESE COMMERCIAL EXPERIMENTAL ADHESIVE AS COMPARED TO THE TOUGH ADHESIVES. LET ME SEE IF I CAN ACTUALLY GET THIS TO PLAY. THE NEXT EXPERIMENT WAS TO SEE WHETHER OR NOT THE PERFORMANCE WOULD BE LIKE IN MORE OF A REALISTIC SETTING. IN THIS CASE OFTENTIMES WE'LL PUT THINGS INTO A SURGICAL SITE WHERE THERE MIGHT BE BLOOD, SALIVA, OTHER TYPES OF BODY FLUIDS. THIS IS A VIDEO ON PLACE IN SKIN WE'VE COATED WITH BLOOD FIRST, YOU CAN SEE IT'S STILL QUIT ADHERENT. IF WE COMPARE, ADHESION ENERGY WHEN NO BLOOD IS THERE, WE OUTPERFORMED CYANOACRILATE, IF YOU PUT BLOOD ON THAT PERFORMANCE IS INHIBITED BUT THIS SYSTEM THERE'S NO EFFECT, PROBABLY BECAUSE THESE ARE HYDRAGELS TO START WITH, ALMOST ALL WATER, HAVING MORE WATER DOESN'T REALLY MATTER. NOW, TO PUT IT IN CONTEXT OF ADHESION ENERGY AND MATRIX TOUGHNESS, HERE ARE THESE MATERIALS, HERE IS CARTILAGE, CYANOACRYLATE. ADHESION IS SIMILAR TO THAT OF CARTILAGE AND BONE. WE THINK THAT THESE HAVE SPECTACULAR PROPERTIES, WE'RE BEGINNING TO EXPLORE, ONE, ABILITY TO ADHERE IN CONTEXT, IN TERMS OF ADHERING TO TENDON, MUSCLE, BONE, CARTILAGE, MENISCUS, SKIN IN THE ORAL CONTEXT, AND IN THE HEART. I'LL GIVE A FEW EXAMPLES. HERE IS ONE ADHERING TO SKIN. THIS IS A MOUSE WHERE WE'VE CREATED A COUPLE OF INCISIONS, APPLIED ON THE BACK. YOU CAN SEE THE MOUSE RUNNING AROUND A WEEK LATER, YOU CAN SEE THEY STAY ADHERENT, IN THE CHALLENGING CONTEXT WITH A LOT OF MOTION THAT'S CONTINUOUSLY HAPPENING. WE CAN USE THESE AS HEMO STATIC AGENTS, A LIVER RAT MODEL, A PUNCH BIOPSY IN THE LIVER OF THE ANIMAL, LOOK AT BLEEDING WITH APPLICATION OF A HEMO STATIC AGENT, THEY WORK EQUIVALENTLY PREVENTING LOSS OF BLOOD, IN THIS CONTEXT. ALSO LOOKING AT USING THESE IN THE ORAL ENVIRONMENT, I WOULD LOVE TO GET FEEDBACK ON TODAY. WE THINK THESE SHOULD BE USEFUL IN THE CONTEXT OF THE MOUTH, WE'RE NOT SURE WHAT THE FIRST APPLICATION SHOULD BE. THIS GIVES A SENSE OF ONE OF THESE, WE'VE APPLIED TO THE GINGIVA, IT STAYS HIGHLY ADHERENT FOR A SUBSTANTIAL PERIOD OF TIME, LOOKED AT ADHERENCE TO THE TONGUE, LIPS, WE'D LIKE TO FIND APPLICATION IN THE MOUTH FOR THESE MATERIALS. I'M GOING BACK TO THE STARTING POINT WITH MY EARLY SLIDES, AND SHOW YOU WHAT IS THE MOST DEMANDING ENVIRONMENT THAT WE'VE LOOKED TO DATE, GOING BACK TO THIS BEATING PIG HEART THAT WE'VE COATED WITH BLOOD TO SEE IF WE COULD GET IT TO ADHERE IN THIS CONTEXT THAT MIGHT MIMIC AN INTRAOPERATIVE SETTING OF USE OF THIS MATERIAL AND SO WE'VE APPLIED IT, COATED IT, PUT IT ON THE BEATING HEART TO ADHERE. THIS PART DOWN HERE, WELL, JUST THE END OF IT WAS ADHERED. WE DIDN'T ADHERE THIS PART, WHY YOU SEE IT PULLING UP. YOU CAN SEE WE'RE PULLING IT BUT THE REGION DOWN HERE IS STAYING ADHERENT. FOR THOSE WHO LIKE TO LOOK ONLINE, THIS IS SOMETHING MY STUDENTS TAUGHT ME, A BARCODE IF YOU SHINE YOUR CELL PHONE, THE CAMERA ON IT, IT WILL ASK YOU IF YOU WANT TO GO TO THE WEB PAGE WITH INFORMATION ON THESE MATERIALS, I'M TRYING TO GET MORE IN TUNE WITH THESE THINGS. I'M GOING TO STOP HERE AND HOPEFULLY I'VE GIVEN YOU A FLAVOR OF HOW WE'RE EXPLORING THE ROLE OF MECHANICAL CUES IN STEM CELL BIOLOGY AND REGENERATIVE MEDICINE, LOOKING AT INTRINSIC MECHANICAL INTERACTION BETWEEN CELL AND ENVIRONMENT AS WELL AS FORCES WE IMPOSE UPON THE CELLS IN TISSUES, AND HOPEFULLY HAVE ALSO PARTICULARLY FOR TRAINEES IN THE AUDIENCE GIVEN YOU A SENSE OF HOW SOMETIMES WHERE YOU END UP IS NOT WHERE YOU INTENDED TO GO, BUT IF YOU KIND OF CONTINUE TO THINK ABOUT THINGS, SOMETIMES YOU REALIZE THERE MIGHT BE INTERESTING CONNECTIONS, THERE MIGHT BE SOME DISCOVERIES IN AREAS YOU DIDN'T ORIGINALLY ANTICIPATE. THANK YOU FOR YOUR TIME AND ATTENTION. I WOULD BE HAPPY TO TAKE ANY QUESTIONS. [APPLAUSE] YOU'RE MAINLY A SURGICAL AUDIENCE, I'LL STILL TRY TO GO OFF THAT SLIDE. YEP? >> CAN YOU IMPREGNATE YOUR GEL -- >> MICROPHONE FOR THE VIDEOCAST. >> SORRY. >> YOU GAVE SOME REDEEMING QUALITIES TO SLUGS, I APPRECIATE THAT. THE FACT THEY CHEW UP MY GARDEN MAKES THEM NOT REDEEMABLE. >> IT WAS A GREAT TALK. EVERYTHING WAS FANTASTIC. I'M REALLY INTERESTED IN YOUR ADHESIVE. I THINK THERE ARE A LOT OF APPLICATIONS YOU'RE NOT GOING TO HAVE A PROBLEM FINDING PEOPLE TO -- BUT TWO THINGS. CAN YOU MODULATE HOWED A HERE ADHERENT THEY ARE, I WOULD LIKE 100 VERSUS 1,000, PULL IT OFF IN A WAY, BECAUSE IF YOU WERE TO ATTACH TO BUCCAL MUCOSA, YOU COULD NEED THAT. TOO HIGH MAY LIFT OFF SOME MUCOSA. TWO, CAN YOU IMPREGNATE THEM WITH DRUGS, DRUG-ELUTING PATCH, EASY APPLICATIONS FOR THAT. >> THE FIRST ONE, YES, WE'VE FOCUSED ON MAKING IT ADHERENT AS POSSIBLE TO SEE HOW FAR TO TAKE THIS. FOR SOME SITUATIONS THAT'S BEEN -- THAT'S BEEN LIMITING. YOU CAN'T GET STRONG ENOUGH ADHESION. YES, WE CAN TUNE THAT JUST BY, FOR EXAMPLE, I DIDN'T SHOW THIS, BY CHANGING THE COMPONENTS OF THE BULK OF THE GEL. WE CAN HAVE A BIG IMPACT. ALSO BY CONTROLLING THE SURFACE CHEMISTRY AS WELL. WE HAVE TWO BASICALLY HANDLES WE CAN TURN FOR THAT. IN TERMS OF THE AREA, WE'RE INTERESTED IN USING THIS AS A LOCAL DRUG DELIVERY SYSTEM. WE CAN IMPREGNATE WITH DRUGS, LOCAL AND SUSTAINED RELEASE. THIS IS THE SPACE IN DENTISTRY I'M INTERESTED IN, INTERESTING APPLICATIONS WHETHER YOU WANT TO HAVE A DRUG THAT YOU COULD PUT IN A PATCH, IN THE MOUTH, AND HAVE IT STICK THERE, BASICALLY DELIVER THAT DRUG TO THAT SITE FOR A FEW DAYS OR A WEEK. AND THEN YOU EITHER PULL THE PATCH OFF OR IT FALLS OFF AND YOU SWALLOW IT. >> (INAUDIBLE). >> JACKIE MAY BE IN THE AUDIENCE. SHE HAD A CLINICAL TRIAL WHERE SHE -- ONE OF THE BIGGEST ISSUES FOR HER, SHE STUDIES -- (INAUDIBLE). ONE OF THE BIGGEST ISSUES, THE MEDICATIONS THAT WERE AVAILABLE TO SLIDE OFF AND NOT COAT THE MOUTH. >> OKAY. >> THEY HAVE LESIONS THROUGHOUT THE MOUTH THAT ARE EXTREMELY PAINFUL. >> OKAY. >> BUT SHE HAD TO FIND VARIOUS FORMULATIONS FOR THINGS TO COAT THE MOUTH PROPERLY TO BE EFFECTIVE. IF SHE WAS HERE -- I DON'T SEE HER. SHE WOULD BE SOMEBODY WHO WOULD HAVE AN APPLICATION IMMEDIATELY. >> OKAY. >> ONE OF THE THINGS -- >> I HAD A QUESTION ABOUT THE EXTERNAL MECHANICAL SYSTEM WITH DISSIPATION OF NEUTROPHILS, WONDER FIGURE YOU LOOKED AT MARKERS OF CELL STRENGTH AND DEATH LIKE ARE CELLS DYING, WHERE ARE NEUTROPHILS GOING? >> WE'VE LOOKED A LITTLE BIT. WE'RE INTERESTED WHETHER THEY ARE GOING OTHER PLACES. SOME DATA, FOR EXAMPLE, THEY MAY GO BACK TO BONE MARROW. I THINK WE'RE NOT SURE, WE DO NOT SEE INCREASED NUMBERS OF THEM, LET'S SAY, IN THE BONE MARROW, OTHER PLACES PEOPLE IDENTIFIED THEY MAY BE GOING AND DOING INTERESTING THINGS, WE'RE NOT SURE AT THIS POINT IN TIME, WE KNOW THEY ARE DISAPPEARING FROM THE TISSUE SITE. >> THANKS. >> YEAH. >> I WOULD LIKE TO ASK, PEOPLE WORKING WITH TISSUE REGENERATION, FOR EXAMPLE SUCCESSFUL TO USE A SCAFFOLD, NATURAL SCAFFOLD THAT DOESN'T CAUSE ANY IMMUNOREACTION, AND THEN EVEN WITH PRINTER, DIFFERENTIATE THE CELLS, DIFFERENT KIND OF CELLS, AND I THINK TO RECONSTRUCT AN ORGAN. COMPARED TO YOUR SYSTEM THAT BASICALLY YOU TRY TO MAKE THE PHYSICAL ENVIRONMENT TO TREAT DIFFERENTIATION, EVENTUALLY MAINTAINMENT OF THE KIND OF CELLS. WHICH KIND OF DIFFERENCE YOU SEE IF YOU DO, I DON'T KNOW, YOU DID THE DNA-SEQ OR I DON'T KNOW, SECRETION OF HORMONES. DID YOU EVER NOTE THIS KIND OF COMPARISON? >> I THINK YOU'RE ASKING WHEN WOULD YOU WANT TO TAKE THE APPROACH TO BUILD TISSUE OR ORGAN OUTSIDE THE BODY AND TRANSPLANT IT VERSUS TRYING TO INDUCE REGENERATION WITH A CUE LIKE THE MECHANICAL STIMULATION, IS THAT -- YEAH, THERE'S A WIDE VARIETY OF DIFFERENT SITUATIONS TO PROMOTE REGENERATION, THOSE TWO STRATEGIES, AMONG OTHERS, WILL BE USEFUL IN DIFFERENT CONTEXTS. IF YOU HAVE AN ACUTE NEED FOR A TISSUE, YOU HAVE TO REPLACE THIS TISSUE TODAY, YOU'RE NOT GOING TO HAVE TIME TO WAIT FOR A NATURAL PROCESS TO BE TRIGGERED AND TO RUN ITS COURSE. SO, YOU KNOW, THIS WHOLE IDEA OF LET'S SAY HAVING A HEART IN A BOX, SOMEBODY COMES WITH HEART FAILURE, YOU CAN TRANSPLANT AN ENGINEERED HEART AS AN ORGAN TODAY IS APPEALING, YOU MAY NOT HAVE TIME TO WAIT IN CERTAIN SITUATIONS SO THAT'S ONE. ALSO PROBABLY DEPEND ON MAGNITUDE. IF THERE ARE -- IF YOU HAVE SUCH SUFFICIENT DAMAGE THAT THERE ARE NOT CELLS FROM THE HOST AVAILABLE TO RESPOND TO THE KIND OF CUES WE'RE INTERESTED IN DELIVERING, THEN THE PROCESS WILL NOT WORK WELL. YOU'RE DEPENDENT ON HAVING A CELL POPULATION THAT CAN RESPOND TO WHATEVER CUES ARE AVAILABLE WITH THIS APPROACH I'VE BEEN DESCRIBING. IF THAT'S NOT TRUE YOU'LL PROBABLY NEED TO PROVIDE THOSE, INCLUDING WAYS YOU'RE DESCRIBING. >> THE LAST FIGURE IS BEAUTIFUL, YOU USE TAPE TO EVENTUALLY REPAIR SOMETHING. I WOULD BE SURPRISED IF THE GEL YOU PUT, YOU DON'T HAVE MIGRATION OF CELLS. DO YOU? >> YES, SO, MAYBE ONE THING I'LL PREFACE, WE'VE LOOKED AT BIOCOMPATIBILITY, PLANTING ON HEART, GOING SUB Q, YOU GET A MILD INFLAMMATORY RESPONSE, NOT VERY SIGNIFICANT, THESE ARE DESIGNED, THE ONE I'M SHOWING HERE, DESIGNED TO NOT DEGRADE. SO YOU DON'T GET VERY MUCH CELL INFILTRATION, NO CELL INFILTRATION. WHEN WE USE DEGRADABLE VERSIONS AS IT DEGRADES CELLS WILL INFILTRATE IT. BUT WE'RE NOT AT THIS POINT IN TIME TRYING TO MAKE THESE THAT ENCOURAGE CELL INFILTRATION, JUST THAT THE MATERIAL HAS GONE AND CELLS FILL THE SPACE, WE HAVEN'T BEEN TRYING TO ENCOURAGE CELL INFILTRATION. >> DR. MOONEY, HI. A GREAT LECTURE. I CAN SEE YOU'RE A REALLY GOOD MENTOR. I REALLY APPRECIATE THAT. CONSIDERING YOUR PROPOSAL OF THE IMPORTANCE OF STRETCH ON PROGENITOR FUNCTION WHAT'S YOUR OPINION OF THE ROLE OF THINGS LIKE YOGA OR EXERCISE ON PROGENITOR ACTIVITY? >> YEAH. I THINK THAT -- I'M SURE THERE'S SOME EFFECT. THE CHALLENGE IS THAT THE KINDS OF STRESSES AND STRAINS THAT GET APPLIED ARE VERY INCONSISTENT, WE DON'T KNOW WHAT THEY ARE. IT'S ACTUALLY -- THERE'S AN INVESTIGATOR AT HARVARD MEDICAL SCHOOL WHO HAS BEEN LOOKING AT THOSE TYPES OF THINGS IN RODENT MODELS, TALKING ABOUT COLLABORATING THAT THE SOFT ROBOTIC SYSTEMS -- >> SHE JUST JOINED OUR INSTITUTE. >> OH, YEAH, OKAY. >> THAT'S WHY I'M CURIOUS. SHE'S MY NEW COLLEAGUE. >> OKAY, YEAH. THIS IS A WAY OF APPLYING VERY STANDARDIZED AND REPRODUCIBLE MECHANICAL CUES, WHICH MAY HELP YOU TO DELINEATE WHAT'S HAPPENING IN THOSE KINDS OF SETTINGS. >> THANK YOU VERY MUCH. >> YEP. >> I HAVE A BUNCH OF QUESTIONS, MAYBE I'LL ASK ONE NOW. JUST FOR THE FUN OF IT, I'VE ALWAYS LOVED THAT SLUG AND THE ADHESION. ONE OF THE -- THINKING ABOUT THIS IN A VARIETY OF AREAS, ORAL CAVITY AND PAIN, AND RIGHT NOW WITH THE HEAL INITIATIVE, AND REGULATING PAIN IN THE LOCAL AREAS, SO ONE OF THE THINGS THAT PERIDONTISTS DO IS GRAFTS, IT'S PAINFUL. WONDER IF YOU COULD HAVE ADHESIVE FACTOR THAT RELEASES LOCAL REGULATION OF PAIN. >> OH. YES, I THINK THAT SHOULD BE VERY FEASIBLE. WE'VE NOT EXPLORED THAT AT ALL. FOR A LOT OF THE LAB, THERE'S DRUG DELIVERY. EACH-- THE SYSTEM, YOU HAVE TO CUSTOMIZE THE SYSTEM FOR EACH DRUG IN TERMS OF CHEMICAL AND PHYSICAL PROPERTIES, EVERY MOLECULE WE'VE TRIED TO HAVE SUSTAINED RELEASE, WE CAN, WE HAVE TO DO A LITTLE BIT OF WORK. WE PROBABLY COULD. JUST NEED TO FIGURE OUT HOW TO DO IT FOR A PARTICULAR MOLECULE. THAT WOULD BE VERY INTERESTING, LOCAL PAIN CONTROL. [APPLAUSE]