Archive for the ‘Tissue Engineering’ Category

3-D Thyroid Bioprinted

Curator: Larry H Bernstein, MD, FCAP

3D-Printed Thyroid Gland



A Russian company announced a successful experiment implanting 3D-printed thyroid glands into mice, and the results will be published next week, said Dmitri Fadin, development director at 3D Printing Solutions.

“We had some difficulties during the study, but in the end the thyroid gland turned out to be functional,” Mr. Fadin told RBTH.

3D Bioprinting Solutions printed the thyroid gland – or to be exact, the gland’s organ construct – in March of this year. At that time, scientific laboratories were saying that they will start printing human thyroid glands if the experiment is successful.

3D Bioprinting Solutions uses existing 3D print technology that makes items from plastic, ceramic and metals, but it had to make adaptations for biological material, that is, for cells. Before transplanting the artificial gland, scientists “carved out” a thyroid in the mice using radioactive iodine.

Vladimir Mironov founded 3D Bioprinting Solutions in 2013. He a tissue engineer, and co-founder of two startups in the U.S., Cardiovascular Tissue Technology, and Cuspis.

Source: rbth.com

Organ printing

3D Biopr

“We had some difficulties during the study, but in the end the thyroid gland turned out to be functional,” Mr. Fadin told RBTH.

Printing Solutions printed the thyroid gland in March 2015



Russian scientists successfully implant the first 3D-printed thyroid gland

A thyroid gland printed last March by 3D Printing Solutions is now proven to be fully functional, and experts say the results will revolutionize medicine.

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Osteo Putty

Larry H. Bernstein, MD, FCAP, Curator



Xtant Medical Announces First Surgical Use Of OsteoSelect® PLUS DBM Putty



Belgrade, Mt (GLOBE NEWSWIRE) – Xtant Medical Holdings, Inc. (NYSE MKT:XTNT), a leader in the development of regenerative medicine products and medical devices, today announced the first surgical implantation of OsteoSelect PLUS Demineralized Bone Matrix (DBM) Putty, developed by its wholly owned subsidiary, Bacterin International, Inc.

OsteoSelect PLUS is a next-generation DBM putty, comprised of OsteoSelect DBM Putty and demineralized cortical chips, designed to provide superior handling for surgeon end users during surgery. Dr. Ali Araghi, DO, Director of the Spine Division at The CORE Institute was the first surgeon to utilize OsteoSelect® PLUS DBM Putty in a spinal fusion procedure.

“OsteoSelect PLUS is aligned with Xtant’s commitment to patient safety and superior clinical outcomes,” stated Dan Goldberger, CEO of Xtant. “OsteoSelect PLUS provides a sterile grafting solution to meet the needs of surgeons and expands our offering in the DBM market space.”

OsteoSelect PLUS was developed in response to surgeon demand. Utilizing surgeon input for design allowed Bacterin to create an additional first class, bone graft substitute in the DBM space and strengthen its comprehensive product portfolio.

About Xtant Medical Holdings
Xtant Medical Holdings, Inc. (NYSE MKT:XTNT) develops, manufactures and markets class-leading regenerative medicine products and medical devices for domestic and international markets. Xtant products serve the specialized needs of orthopedic and neurological surgeons, including orthobiologics for the promotion of bone healing, implants and instrumentation for the treatment of spinal disease, tissue grafts for the treatment of orthopedic disorders, and biologics to promote healing following cranial, and foot and ankle surgeries. With core competencies in both biologic and non-biologic surgical technologies, Xtant can leverage its resources to successfully compete in global neurological and orthopedic surgery markets. For further information, please visit http://www.xtantmedical.com.

Important Cautions Regarding Forward-looking Statements
This press release contains certain disclosures that may be deemed forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995 that are subject to significant risks and uncertainties. Forward-looking statements include statements that are predictive in nature, that depend upon or refer to future events or conditions, or that include words such as “continue,” “efforts,” “expects,” “anticipates,” “intends,” “plans,” “believes,” “estimates,” “projects,” “forecasts,” “strategy,” “will,” “goal,” “target,” “prospects,” “potential,” “optimistic,” “confident,” “likely,” “probable” or similar expressions or the negative thereof. Statements of historical fact also may be deemed to be forward-looking statements. We caution that these statements by their nature involve risks and uncertainties, and actual results may differ materially depending on a variety of important factors, including, among others: the Company’s ability to successfully integrate the acquisition of X-spine; the ability of the Company’s sales force to achieve expected results; the Company’s ability to meet its existing and anticipated contractual obligations, including financial covenant and other obligations contained in the Company’s secured lending facility; the Company’s ability to manage cash flow; the Company’s ability to develop, market, sell and distribute desirable applications, products and services and to protect its intellectual property; the ability of the Company’s customers to pay and the timeliness of such payments; the Company’s ability to obtain financing as and when needed; changes in consumer demands and preferences; the Company’s ability to attract and retain management and employees with appropriate skills and expertise; the impact of changes in market, legal and regulatory conditions and in the applicable business environment, including actions of competitors; and other factors. Additional risk factors are listed in the Company’s Annual Report on Form 10-K and Quarterly Reports on Form 10-Q under the heading “Risk Factors.” The Company undertakes no obligation to release publicly any revisions to any forward-looking statements to reflect events or circumstances after the date hereof or to reflect the occurrence of unanticipated events, except as required by law.

© Copyright 2015, GlobeNewswire, Inc. All Rights Reserved.

SOURCE: Xtant Medical Holdings, Inc.

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FDA Cellular & Gene Therapy Guidances: Implications for CRSPR/Cas9 Trials

Reporter: Stephen J. Williams, PhD

The recent announcement by Editas CEO Katrine Bosley to pursue a CRSPR/Cas9 gene therapy trial to correct defects in an yet to be disclosed gene to treat one form of a rare eye disease called Leber congenital amaurosis (multiple mutant genes have been linked to the disease) have put an interesting emphasis on the need for a regulatory framework to initiate these trials. Indeed at the 2015 EmTechMIT Conference Editas CEO Katrine Bosley had mentioned this particular issue: the need for discourse with FDA and regulatory bodies to establish guidelines for design of clinical trials using the CRSPR gene editing tool.

See the LIVE NOTES from Editas CEO Katrine Bosley on using CRSPR as a gene therapy from the 2015 EmTechMIT Conference at https://pharmaceuticalintelligence.com/2015/11/03/live-1132015-130pm-the-15th-annual-emtech-mit-mit-media-lab-top-10-breakthrough-technologies-2015-innovators-under-35/

To this effect, I have listed below, the multiple FDA Guidance Documents surrounding gene therapy to show that, in the past year, the FDA has shown great commitment to devise a regulatory framework for this therapeutic area.

Cellular & Gene Therapy Guidance Documents

Withdrawn Guidance Documents

Three other posts on this site goes into detail into three of the above-mentioned Guidance Documents

FDA Guidance on Use of Xenotransplanted Products in Human: Implications in 3D Printing

New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue

FDA Guidance Documents Update Nov. 2015 on Devices, Animal Studies, Gene Therapy, Liposomes


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New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue

Reporter: Stephen J. Williams, Ph.D.

The FDA recently came out with a Draft Guidance on use of human cells, tissues and cellular and tissue-based products (HCT/P) {defined in 21 CFR 1271.3(d)} and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.

A full copy of the PDF can be found here for reference but the following is a summary of points of the guidance.FO508ver – 2015-373 HomologousUseGuidanceFinal102715

In 21 CFR 1271.10, the regulations identify the criteria for regulation solely under section 361 of the PHS Act and 21 CFR Part 1271. An HCT/P is regulated solely under section 361 of the PHS Act and 21 CFR Part 1271 if it meets all of the following criteria (21 CFR 1271.10(a)):

  • The HCT/P is minimally manipulated;
  • The HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent;
  • The manufacture of the HCT/P does not involve the combination of the cells or tissues with another article, except for water, crystalloids, or a sterilizing, preserving, or storage agent, provided that the addition of water, crystalloids, or the sterilizing, preserving, or storage agent does not raise new clinical safety concerns with respect to the HCT/P; and
  • Either:
  1. The HCT/P does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function; or
  2. The HCT/P has a systemic effect or is dependent upon the metabolic activity of living cells for its primary function, and:
  3. Is for autologous use;
  4. Is for allogeneic use in a first-degree or second-degree blood relative; or
  5. Is for reproductive use.

If an HCT/P does not meet all of the criteria in 21 CFR 1271.10(a), and the establishment that manufactures the HCT/P does not qualify for any of the exceptions in 21 CFR 1271.15, the HCT/P will be regulated as a drug, device, and/or biological product under the Federal Food, Drug and Cosmetic Act (FD&C Act), and/or section 351 of the PHS Act, and applicable regulations, including 21 CFR Part 1271, and pre-market review will be required.

1 Examples of HCT/Ps include, but are not limited to, bone, ligament, skin, dura mater, heart valve, cornea, hematopoietic stem/progenitor cells derived from peripheral and cord blood, manipulated autologous chondrocytes, epithelial cells on a synthetic matrix, and semen or other reproductive tissue. The following articles are not considered HCT/Ps: (1) Vascularized human organs for transplantation; (2) Whole blood or blood components or blood derivative products subject to listing under 21 CFR Parts 607 and 207, respectively; (3) Secreted or extracted human products, such as milk, collagen, and cell factors, except that semen is considered an HCT/P; (4) Minimally manipulated bone marrow for homologous use and not combined with another article (except for water, crystalloids, or a sterilizing, preserving, or storage agent, if the addition of the agent does not raise new clinical safety concerns with respect to the bone marrow); (5) Ancillary products used in the manufacture of HCT/P; (6) Cells, tissues, and organs derived from animals other than humans; (7) In vitro diagnostic products as defined in 21 CFR 809.3(a); and (8) Blood vessels recovered with an organ, as defined in 42 CFR 121.2 that are intended for use in organ transplantation and labeled “For use in organ transplantation only.” (21 CFR 1271.3(d))

Contains Nonbinding Recommendations
Draft – Not for Implementation

Section 1271.10(a)(2) (21 CFR 1271.10(a)(2)) provides that one of the criteria for an HCT/P to be regulated solely under section 361 of the PHS Act is that the “HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.” As defined in 21 CFR 1271.3(c), homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor. This criterion reflects the Agency’s conclusion that there would be increased safety and effectiveness concerns for HCT/Ps that are intended for a non-homologous use, because there is less basis on which to predict the product’s behavior, whereas HCT/Ps for homologous use can reasonably be expected to function appropriately (assuming all of the other criteria are also met).2 In applying the homologous use criterion, FDA will determine what the intended use of the HCT/P is, as reflected by the the labeling, advertising, and other indications of a manufacturer’s objective intent, and will then apply the homologous use definition.

FDA has received many inquiries from manufacturers about whether their HCT/Ps meet the homologous use criterion in 21 CFR 1271.10(a)(2). Additionally, transplant and healthcare providers often need to know this information about the HCT/Ps that they are considering for use in their patients. This guidance provides examples of different types of HCT/Ps and how the regulation in 21 CFR 1271.10(a)(2) applies to them, and provides general principles that can be applied to HCT/Ps that may be developed in the future. In some of the examples, the HCT/Ps may fail to meet more than one of the four criteria in 21 CFR 1271.10(a).


  1. What is the definition of homologous use?

Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor (21 CFR 1271.3(c)), including when such cells or tissues are for autologous use. We generally consider an HCT/P to be for homologous use when it is used to repair, reconstruct, replace, or supplement:

  • Recipient cells or tissues that are identical (e.g., skin for skin) to the donor cells or tissues, and perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor; or,
  • Recipient cells that may not be identical to the donor’s cells, or recipient tissues that may not be identical to the donor’s tissues, but that perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor.3

2 Proposed Approach to Regulation of Cellular and Tissue-Based Products, FDA Docket. No. 97N-0068 (February. 28, 1997) page 19. http://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/tissue/ ucm062601.pdf.

3“Establishment Registration and Listing for Manufacturers of Human Cellular and Tissue-Based Products” 63 FR 26744 at 26749 (May 14, 1998).

Contains Nonbinding Recommendations
Draft – Not for Implementation

1-1. A heart valve is transplanted to replace a dysfunctional heart valve. This is homologous use because the donor heart valve performs the same basic function in the donor as in the recipient of ensuring unidirectional blood flow within the heart.

1-2. Pericardium is intended to be used as a wound covering for dura mater defects. This is homologous use because the pericardium is intended to repair or reconstruct the dura mater and serve as a covering in the recipient, which is one of the basic functions it performs in the donor.

Generally, if an HCT/P is intended for use as an unproven treatment for a myriad of

diseases or conditions, the HCT/P is likely not intended for homologous use only.4

  1. What does FDA mean by repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues?

Repair generally means the physical or mechanical restoration of tissues, including by covering or protecting. For example, FDA generally would consider skin removed from a donor and then transplanted to a recipient in order to cover a burn wound to be a homologous use. Reconstruction generally means surgical reassembling or re-forming. For example, reconstruction generally would include the reestablishment of the physical integrity of a damaged aorta.5 Replacement generally means substitution of a missing tissue or cell, for example, the replacement of a damaged or diseased cornea with a healthy cornea or the replacement of donor hematopoietic stem/progenitor cells in a recipient with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. Supplementation generally means to add to, or complete. For example, FDA generally would consider homologous uses to be the implantation of dermal matrix into the facial wrinkles to supplement a recipient’s tissues and the use of bone chips to supplement bony defects. Repair, reconstruction, replacement, and supplementation are not mutually exclusive functions and an HCT/P could perform more than one of these functions for a given intended use.

  1. What does FDA mean by “the same basic function or functions” in the definition of homologous use?

For the purpose of applying the regulatory framework, the same basic function or functions of HCT/Ps are considered to be those basic functions the HCT/P performs in the body of the donor, which, when transplanted, implanted, infused, or transferred, the HCT/P would be expected to perform in the recipient. It is not necessary for the HCT/P in the recipient to perform all of the basic functions it performed in the donor, in order to

4 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).

5 “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-Based Product Establishments; Inspection and Enforcement” 69 FR 68612 at 68643 (November 24, 2004) states, “HCT/Ps with claims for “reconstruction or repair” can be regulated solely under section 361 of the PHS Act, provided the HCT/P meets all the criteria in § 1271.10, including minimal manipulation and homologous use.”

Contains Nonbinding Recommendations
Draft – Not for Implementation

meet the definition of homologous use. However, to meet the definition of homologous use, any of the basic functions that the HCT/P is expected to perform in the recipient must be a basic function that the HCT/P performed in the donor.

A homologous use for a structural tissue would generally be to perform a structural function in the recipient, for example, to physically support or serve as a barrier or conduit, or connect, cover, or cushion.

A homologous use for a cellular or nonstructural tissue would generally be a metabolic or biochemical function in the recipient, such as, hematopoietic, immune, and endocrine functions.

3-1. The basic functions of hematopoietic stem/progenitor cells (HPCs) include to form and to replenish the hematopoietic system. Sources of HPCs include cord blood, peripheral blood, and bone marrow.6

  1. HPCs derived from peripheral blood are intended for transplantation into an individual with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. This is homologous use because the peripheral blood product performs the same basic function of reconstituting the hematopoietic system in the recipient.
  2. HPCs derived from bone marrow are infused into an artery with a balloon catheter for the purpose of limiting ventricular remodeling following acute myocardial infarction. This is not homologous use because limiting ventricular remodeling is not a basic function of bone marrow.
  3. A manufacturer provides HPCs derived from cord blood with a package insert stating that cord blood may be infused intravenously to differentiate into neuronal cells for treatment of cerebral palsy. This is not homologous use because there is insufficient evidence to support that such differentiation is a basic function of these cells in the donor.

3-2. The basic functions of the cornea include protecting the eye by forming its outermost layer and serving as the refracting medium of the eye. A corneal graft is transplanted to restore sight in a patient with corneal blindness. This is homologous use because a corneal graft performs the same basic functions in the donor as in the recipient.

3-3. The basic functions of a vein or artery include serving as a conduit for blood flow throughout the body. A cryopreserved vein or artery is used for arteriovenous access during hemodialysis. This is homologous use because the vein or artery is supplementing the vessel as a conduit for blood flow.

3-4. The basic functions of amniotic membrane include covering, protecting, serving as a selective barrier for the movement of nutrients between the external and in utero

6 Bone marrow meets the definition of an HCT/P only if is it more than minimally manipulated; intended by the manufacturer for a non-homologous use, or combined with certain drugs or devices.

Contains Nonbinding Recommendations
Draft – Not for Implementation

environment, and to retain fluid in utero. Amniotic membrane is used for bone tissue replacement to support bone regeneration following surgery to repair or replace bone defects. This is not a homologous use because bone regeneration is not a basic function of amniotic membrane.

3-5. The basic functions of pericardium include covering, protecting against infection, fixing the heart to the mediastinum, and providing lubrication to allow normal heart movement within chest. Autologous pericardium is used to replace a dysfunctional heart valve in the same patient. This is not homologous use because facilitating unidirectional blood flow is not a basic function of pericardium.

  1. Does my HCT/P have to be used in the same anatomic location to perform the same basic function or functions?

An HCT/P may perform the same basic function or functions even when it is not used in the same anatomic location where it existed in the donor.7 A transplanted HCT/P could replace missing tissue, or repair, reconstruct, or supplement tissue that is missing or damaged, either when placed in the same or different anatomic location, as long as it performs the same basic function(s) in the recipient as in the donor.

4-1. The basic functions of skin include covering, protecting the body from external force, and serving as a water-resistant barrier to pathogens or other damaging agents in the external environment. The dermis is the elastic connective tissue layer of the skin that provides a supportive layer of the integument and protects the body from mechanical stress.

  1. An acellular dermal product is used for supplemental support, protection, reinforcement, or covering for a tendon. This is homologous use because in both anatomic locations, the dermis provides support and protects the soft tissue structure from mechanical stress.
  2. An acellular dermal product is used for tendon replacement or repair. This is not homologous use because serving as a connection between muscle and bone is not a basic function of dermis.

4-2. The basic functions of amniotic membrane include serving as a selective barrier for the movement of nutrients between the external and in utero environment and to retain fluid in utero. An amniotic membrane product is used for wound healing of dermal ulcers and defects. This is not homologous use because wound healing of dermal lesions is not a basic function of amniotic membrane.

4-3. The basic functions of pancreatic islets include regulating glucose homeostasis within the body. Pancreatic islets are transplanted into the liver through the portal vein,

7 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).


Contains Nonbinding Recommendations
Draft – Not for Implementation

for preservation of endocrine function after pancreatectomy. This is homologous use because the regulation of glucose homeostasis is a basic function of pancreatic islets.

  1. What does FDA mean by “intended for homologous use” in 21 CFR 1271.10(a)(2)?

The regulatory criterion in 21 CFR 1271.10(a)(2) states that the HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.

Labeling includes the HCT/P label and any written, printed, or graphic materials that supplement, explain, or are textually related to the product, and which are disseminated by or on behalf of its manufacturer.8 Advertising includes information, other than labeling, that originates from the same source as the product and that is intended to supplement, explain, or be textually related to the product (e.g., print advertising, broadcast advertising, electronic advertising (including the Internet), statements of company representatives).9

An HCT/P is intended for homologous use when its labeling, advertising, or other indications of the manufacturer’s objective intent refer to only homologous uses for the HCT/P. When an HCT/P’s labeling, advertising, or other indications of the manufacturer’s objective intent refer to non-homologous uses, the HCT/P would not meet the homologous use criterion in 21 CFR 1271.10(a)(2).

  1. What does FDA mean by “manufacturer’s objective intent” in 21 CFR 1271.10(a)(2)?

A manufacturer’s objective intent is determined by the expressions of the manufacturer or its representatives, or may be shown by the circumstances surrounding the distribution of the article. A manufacturer’s objective intent may, for example, be shown by labeling claims, advertising matter, or oral or written statements by the manufacturer or its representatives. It may be shown by the circumstances that the HCT/P is, with the knowledge of the manufacturer or its representatives, offered for a purpose for which it is neither labeled nor advertised.

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3D BioPrinted Carbon Nanotubes used to Stimulate Bone Regrowth

Reporter: Irina Robu, PhD

Bone disorders are of significant concern due to increase in the median age of our population and at this present time bone grafts have are used to restore damaged bone. However, synthetic biomaterials are now being used as bone graft substitutes and they are selected for structural restoration based on their biomechanical properties. Lately, scaffolds are engineered to be bioactive to enhance tissue growth. These scaffolds are usually porous, made of biodegradable factors, drugs or stem cells.

The research group led by Dr. Maria Vallet-Regi at Faculty of Pharmacy-Universidad Complutense de Madrid showed that carbon nanotubes to the mix to create 3D electrical network within the bone tissue can stimulate bone cell regrowth. The polymer they used was polycarpolactone (PCL), which is rather easy to 3D print.
According to Mercedes Vila, the Principal Investigator in charge of the project, the carbon nanotubes were added to the bio-printable material mixture to create a three-dimensional electrical conducting network all through the volume of the scaffold, which would allow the application of this stimulation to the scaffold once implanted on the damaged bone site.
“In this sense, electrical stimulation has been explored since the discovery of the presence of electrical potentials in mechanically loaded bones,” Mercedes pointed out. “Certain types of cell behavior, such as adhesion and differentiation, can be affected by the application of electrical stimulation. Thus, the creation of a permanent charge on the material surface, positive or negative, as well as a direct electrical stimulation can promote the attraction of charged ions from the environment to the cells. This would modify their protein adsorption with the subsequent influence on the cells’ metabolic activity. Therefore, the use of electrical stimulation after biomaterial implantation to favor cell adhesion and differentiation and, consequently, induce bone healing seems a smart approach to accelerate the osteointegration process.”

Adding CNTs into the bio-printed polymer and mineral prosthetic bone can stimulate regrowth of the actual bone cells. However, bio-printing CNTs created no extra difficulties, as they are so thin that they can be extruded with ease through any pneumatic syringe. Most of the complications are related to finding the correct viscosity in the combination of CPL and hydroxypatite.

“Finding the right right viscosity to be extruded through the syringe while keeping enough robustness to get the 3D scaffold printed at room temperature, was complicated,” Mercedes admitted. “At the same time as the slurry was prepared in dichloromethane solution for diluting the PCL, achieving the right viscosity while evaporating the solvent was tricky. Moreover, once the PCL and the hydroxyapatite were mixed together, the addition of the CNTs was performed and reaching a proper dispersion took a bit of stirring time.”

Using EnvisionTEC’s 3D bioplotter, the researchers were able to create very complex 3D structures which would enhance the future for tissue replacements as it allows tailored solutions by capturing the anatomical information of the patient’s wound by computed tomography and magnetic resonance, for example, to obtain a personalized and unique implant.

As with many other 3D printing applications, it appears we are only starting to scratch the surface of the possibilities that are ahead for bioprinting.



Other related articles published in this Open Access Online Scientific Journal include the following:

search for Bone related articles, please place here references

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Gene Editing by creation of a complement without transcription error

Larry H. Bernstein, MD, FCAP, Curator


Nanoparticle-Based Artificial Transcription Factor  

NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation

Abstract Image

Transcription factor (TF) proteins are master regulators of transcriptional activity and gene expression. TF-based gene regulation is a promising approach for many biological applications; however, several limitations hinder the full potential of TFs. Herein, we developed an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. We demonstrate that NanoScript localizes within the nucleus and initiates transcription of a reporter plasmid by over 15-fold. Moreover, NanoScript can effectively transcribe targeted genes on endogenous DNA in a nonviral manner. Because NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform, it has great potential for various stem cell applications.




  • Transcription Factors (TF) are proteins that regulate transcription and gene expression
  • NanoScript is an versatile, nanoparticle-based platform that mimics TF structure and biological function
  • NanoScript is stable in physiological environments and localizes within the nucleus
  • NanoScript initiates targeted gene expression by over 15-fold to 30 fold, which would be critical for stem cell differentiation and cellular reprogramming
  • NanoScript transcribes endogenous genes on native DNA in a non-viral manner

Transcription factor (TF) proteins are master regulators of transcriptional activity and gene expression. TF-based gene regulation is an essential approach for many biological applications such as stem cell differentiation and cellular programming, however, several limitations hinder the full potential of TFs.

To address this challenge, researchers in Prof. KiBum Lee’s group (Sahishnu Patel and Perry Yin) developed an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. They demonstrated that NanoScript localizes within the nucleus and initiates transcription of a targeted gene with high efficiency. Moreover, NanoScript can effectively transcribe targeted genes on endogenous DNA in a non-viral manner.

NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform. NanoScript has two attractive features that make this the perfect platform for stem cell-based application. First, because gene regulation by NanoScript is non-viral, it serves as an attractive alternative to current differentiation methods that use viral vectors. Second, by simply rearranging the sequence of one molecule on NanoScript, NanoScript can target any differentiation-specific genes and induce differentiation, and thus has excellent prospect for applications in stem cell biology and cellular reprogramming.

Perry To-tien Yin
PhD Candidate, Rutgers University
Prospects for graphene–nanoparticle-based hybrid sensors

PT Yin, TH Kim, JW Choi, KB Lee
Physical Chemistry Chemical Physics 15 (31), 12785-12799
31 2013
Axonal Alignment and Enhanced Neuronal Differentiation of Neural Stem Cells on Graphene‐Nanoparticle Hybrid Structures

A Solanki, STD Chueng, PT Yin, R Kappera, M Chhowalla, KB Lee
Advanced Materials 25 (38), 5477-5482
22 2013
Label‐Free Polypeptide‐Based Enzyme Detection Using a Graphene‐Nanoparticle Hybrid Sensor

S Myung, PT Yin, C Kim, J Park, A Solanki, PI Reyes, Y Lu, KS Kim, …
Advanced Materials 24 (45), 6081-6087
22 2012
Guiding Stem Cell Differentiation into Oligodendrocytes Using Graphene‐Nanofiber Hybrid Scaffolds

S Shah, PT Yin, TM Uehara, STD Chueng, L Yang, KB Lee
Advanced materials 26 (22), 3673-3680
21 2014
Design, Synthesis, and Characterization of Graphene–Nanoparticle Hybrid Materials for Bioapplications

PT Yin, S Shah, M Chhowalla, KB Lee
Chemical reviews 115 (7), 2483-2531
16 2015
Multimodal Magnetic Core–Shell Nanoparticles for Effective Stem‐Cell Differentiation and Imaging

B Shah, PT Yin, S Ghoshal, KB Lee
Angewandte Chemie 125 (24), 6310-6315
16 2013
Nanotopography-mediated reverse uptake for siRNA delivery into neural stem cells to enhance neuronal differentiation

A Solanki, S Shah, PT Yin, KB Lee
Scientific reports 3
14 2013
Combined Magnetic Nanoparticle‐based MicroRNA and Hyperthermia Therapy to Enhance Apoptosis in Brain Cancer Cells

PT Yin, BP Shah, KB Lee
small 10 (20), 4106-4112
11 2014

A highly robust, efficient nanoparticle-based platform to advance stem cell therapeutics

(Nanowerk News) Associate Professor Ki-Bum Lee has developed patent-pending technology that may overcome one of the critical barriers to harnessing the full therapeutic potential of stem cells.
One of the major challenges facing researchers interested in regenerating cells and growing new tissue to treat debilitating injuries and diseases such as Parkinson’s disease, heart disease, and spinal cord trauma, is creating an easy, effective, and non-toxic methodology to control differentiation into specific cell lineages. Lee and colleagues at Rutgers and Kyoto University in Japan have invented a platform they call NanoScript, an important breakthrough for researchers in the area of gene expression. Gene expression is the way information encoded in a gene is used to direct the assembly of a protein molecule, which is integral to the process of tissue development through stem cell therapeutics.
Stem cells hold great promise for a wide range of medical therapeutics as they have the ability to grow tissue throughout the body. In many tissues, stem cells have an almost limitless ability to divide and replenish other cells, serving as an internal repair system.

Schematic representation of NanoScript’s design and function. (a) By assembling individual STF molecules, including the DBD (DNA-binding domain), AD (activation domain), and NLS (nuclear localization signal), onto a single 10 nm gold nanoparticle, we have developed the NanoScript platform to replicate the structure and function of TFs. This NanoScript penetrates the cell membrane and enters the nucleus through the nuclear receptor with the help of the NLS peptide. Once in the nucleus, NanoScript interacts with DNA to initiate transcriptional activity and induce gene expression. (b) When comparing the structure of NanoScript to representative TF proteins, the three essential domains are effectively replicated. The linker domain (LD) fuses the multidomain protein together and is replicated by the gold nanoparticle (AuNP). (c) The DBD binds to complementary DNA sequences, while the AD recruits transcriptional machinery components such as RNA polymerase II (RNA Pol II), mediator complex, and general transcription factors (GTFs). The synergistic function of the DBD and AD moieties on NanoScript initiates transcriptional activity and expression of targeted genes. (d) The AuNPs are monodisperse and uniform. The NanoScript constructs are shown to effectively localize within the nucleus, which is important because transcriptional activity occurs only in the nucleus. (Reprinted with permission y American Chemical Society) (click on image to enlarge)

Read more: Using nanotechnology to regulate gene expression at the transcriptional level

Transcription factor (TF) proteins are master regulators of gene expression. TF proteins play a pivotal role in regulating stem cell differentiation. Although some have tried to make synthetic molecules that perform the functions of natural transcription factors, NanoScript is the first nanomaterial TF protein that can interact with endogenous DNA.
ACS Nano, a publication of the American Chemical Society (ACS), has published Lee’s research on NanoScript (“NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation”). The research is supported by a grant from the National Institutes of Health (NIH).
“Our motivation was to develop a highly robust, efficient nanoparticle-based platform that can regulate gene expression and eventually stem cell differentiation,” said Lee, who leads a Rutgers research group primarily focused on developing and integrating nanotechnology with chemical biology to modulate signaling pathways in cancer and stem cells. “Because NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform, it has great potential to do exactly that. The field of stem cell biology now has another platform to regulate differentiation while the field of nanotechnology has demonstrated for the first time that we can regulate gene expression at the transcriptional level.”
NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles.
“NanoScript localizes within the nucleus and initiates transcription of a reporter plasmid by up to 30-fold,” said Sahishnu Patel, Rutgers Chemistry graduate student and co-author of the ACS Nano publication. “NanoScript can effectively transcribe targeted genes on endogenous DNA in a nonviral manner.”
Lee said the next step for his research is to study what happens to the gold nanoparticles after NanoScript is utilized, to ensure no toxic effects arise, and to ensure the effectiveness of NanoScript over long periods of time.
“Due to the unique tunable properties of NanoScript, we are highly confident this platform not only will serve as a desirable alternative to conventional gene-regulating methods,” Lee said, “but also has direct employment for applications involving gene manipulation such as stem cell differentiation, cancer therapy, and cellular reprogramming. Our research will continue to evaluate the long-term implications for the technology.”
Lee, originally from South Korea, joined the Rutgers faculty in 2008 and has earned many honors including the NIH Director’s New Innovator Award. Lee received his Ph.D. in Chemistry from Northwestern University where he studied with Professor Chad. A. Mirkin, a pioneer in the coupling of nanotechnology and biomolecules. Lee completed his postdoctoral training at The Scripps Research Institute with Professor Peter G. Schultz. Lee has served as a Visiting Scholar at both Princeton University and UCLA Medical School.
The primary interest of Lee’s group is to develop and integrate nanotechnologies and chemical functional genomics to modulate signaling pathways in mammalian cells towards specific cell lineages or behaviors. He has published more than 50 articles and filed for 17 corresponding patents.
Source: Rutgers University

Read more: A highly robust, efficient nanoparticle-based platform to advance stem cell therapeutics

Nanoparticle-based transcription factor mimics


Biologists have been enhancing expression of specific genes with plasmids and viruses for decades, which has been essential to uncovering the function of numerous genes and the relationships among the proteins they encode. However, tools that allow enhancement of expression of endogenous genes at the transcriptional level could be a powerful complement to these strategies. Many chemical biologists have made enormous progress developing molecular tools for this purpose; recent work by a group at Rutgers suggests how nanotechnology might allow application of this strategy in living organisms, and perhaps one day in patients.

In a paper published in ACS Nano, researchers led by KiBum Lee synthesized gold nanoparticles bearing synthetic or shortened versions of the three essential components of transcription factors (TFs), the proteins that “turn on” expression of specific genes in cells. Specifically, polyamides previously designed to bind to a specific promoter sequence, transactivation peptides, and nuclear localization peptides were conjugated to the nanoparticle surface. These nanoparticles enhanced expression of both a reporter plasmid (by ~15-fold) and several endogenous genes (by up to 65%). This enhancement is much greater than that possible using previous constructs lacking nuclear localization sequences; the team incorporated a high proportion of those peptides to ensure efficient delivery to the nucleus.

Nanoscript, a synthetic transciption factor
Diagram of the synthetic TF mimic (termed NanoScript). Decorated particles are ~35 nm in diameter. Letters are amino acid sequences; Py-Im, N-methylpyrrole-N-methylimidazole.

These nanoparticles offer an alternative to delivering protein TFs, which remains extremely challenging despite considerable effort towards the development of delivery systems that transport cargo into cells. Among other barriers to the use of native TFs, incorporating them into polymeric or lipid-based carriers often alters their shape, which would likely reduce their function.

While the group suggests future generations of these nanoparticles might one day be used to treat diseases caused by defects in TF genes, many questions remain. First, the duration of gene expression enhancement is not known; the study only assesses effects at 48 h post-administration. Further, whether gold is the best material for the core remains unclear, as its non-biodegradability means the particles would likely accumulate in the liver over time; synthetic TFs with biodegradable cores might also be considered.

Patel S et al., NanoScript: a nanoparticle-based artificial transcription factor for effective gene regulation,ACS Nano 2014; published online Sep 3.


Biocompatibility and Toxicity of Nanobiomaterials

“Biocompatibility and Toxicity of Nanobiomaterials” is an annual special issue published in “Journal of Nanomaterials.”


Porous Ti6Al4V Scaffold Directly Fabricated by Sintering: Preparation and In Vivo Experiment
Xuesong Zhang, Guoquan Zheng, Jiaqi Wang, Yonggang Zhang, Guoqiang Zhang, Zhongli Li, and Yan Wang
Department of Orthopaedics, Chinese People’s Liberation Army General Hospital, Beijing 100853, China AcademicEditor:XiaomingLi
The interface between the implant and host bone plays a key role in maintaining primary and long-term stability of the implants. Surface modification of implant can enhance bone in growth and increase bone formation to create firm osseo integration between the implant and host bone and reduce the risk of implant losing. This paper mainly focuses on the fabricating of 3-dimensiona interconnected porous titanium by sintering of Ti6Al4V powders, which could be processed to the surface of the implant shaft and was integrated with bone morphogenetic proteins (BMPs). The structure and mechanical property of porous Ti6Al4V was observed and tested. Implant shaft with surface of porous titanium was implanted into the femoral medullary cavity of dog after combining with BMPs. The results showed that the structure and elastic modulus of 3D interconnected porous titanium was similar to cancellous bone; porous titanium combined with BMP was found to have large amount of fibrous tissue with fibroblastic cells; bone formation was significantly greater in 6 weeks postoperatively than in 3 weeks after operation. Porous titanium fabricated by powders sintering and combined with BMPs could induce tissue formation and increase bone formation to create firm osseo integration between the implant and host bone.

Journal of Materials Chemistry B   Issue 39, 2013

Materials for biology and medicine
Synthesis of nanoparticles, their biocompatibility, and toxicity behavior for biomedical applications
J. Mater. Chem. B, 2013,1, 5186-5200    DOI: http://dx.doi.org:/10.1039/C3TB20738B

Nanomaterials research has in part been focused on their use in biomedical applications for more than several decades. However, in recent years this field has been developing to a much more advanced stage by carefully controlling the size, shape, and surface-modification of nanoparticles. This review provides an overview of two classes of nanoparticles, namely iron oxide and NaLnF4, and synthesis methods, characterization techniques, study of biocompatibility, toxicity behavior, and applications of iron oxide nanoparticles and NaLnF4nanoparticles as contrast agents in magnetic resonance imaging. Their optical properties will only briefly be mentioned. Iron oxide nanoparticles show a saturation of magnetization at low field, therefore, the focus will be MLnF4 (Ln = Dy3+, Ho3+, and Gd3+) paramagnetic nanoparticles as alternative contrast agents which can sustain their magnetization at high field. The reason is that more potent contrast agents are needed at magnetic fields higher than 7 T, where most animal MRI is being done these days. Furthermore we observe that the extent of cytotoxicity is not fully understood at present, in part because it is dependent on the size, capping materials, dose of nanoparticles, and surface chemistry, and thus needs optimization of the multidimensional phenomenon. Therefore, it needs further careful investigation before being used in clinical applications.

Graphical abstract: Synthesis of nanoparticles, their biocompatibility, and toxicity behavior for biomedical applications


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HAMLET interacts with lipid membranes and perturbs their structure and integrity

HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal …. of the alternative complement pathway preserves photoreceptors after retinal injury ….. Life-long in vivo cell-lineage tracing shows that no oogenesis originates from …. ananoparticle-based artificial transcription factor for effective gene regulation …

Authors: Ann-Kristin Mossberg, Maja Puchades, Øyvind Halskau, Anne Baumann, Ingela Lanekoff, Yinxia Chao, Aurora Martinez, Catharina Svanborg, & Roger Karlsson



Background – Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure.

Methodology/Principal Findings – We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles.

Conclusions/Significance – The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

Source: Public Library of Science ONE; 5(2) (02/23/10) 

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Scientists at University of Stuttgart, University of Virgnia and Koc Universityhave 3D printed multimaterial parts with multidirectional stiffness gradients. By mixing their expertise in materials engineering and digital processing, scientists create a series of sets of cellulose-based filaments with modifying mechanical and rheological properties, despite having similar compositions. The materials were then used in conjunction with each other to program specific deformation profiles into complex parts.

Functionally graded materials (FGMs) have a gradually changing composition or structure and can be designed to create a precise stiffness profile in each part was then generated. When printed, the samples could be deformed in distinctive profiles due the alteration in stiffness across the geometry of the parts. Eventually, scientists had ‘programmed’ a set of desired deformation geometries.




Three-dimensional printed microfibers used to reinforce hydrogels

Reporter: Irina Robu, PhD

The field of tissue engineering has continued to evolve with the intention to restore, replace and regenerate loss and damaged tissues. Engineered tissues have been able to help millions of people which why their development and design is important. The tree components that are needed for this are cells, scaffold and bioactive factors. 

The scaffold is responsible for providing the structure and support needed to provide tissue development but they differ in composition, design, material properties, structure properties etc. In the spite of everything,  the concept of a scaffold is to mimic the function of the native extracellular matrix by creating similar architectural, biological and mechanical features.

One classic material that is used for tissue engineering are hydrogels, which are designed to provide a hydrated 3D environment of the cells which act as cell carriers. But, hydrogels are unable to provide the needed mechanical properties needed to form extracellular matrix.

Taking the account the limitation, scientists from Medical Center at Utrecht University created a 3D dimensional microfiber network through melt electrospinning to reinforce hydrogel architecture, in order to provide mechanical and biological stable environment for engineered constructs. They took into account that they hydrogel mechanical properties should match those of the target tissue to promote enhanced performance. 

Researchers in the past have tried to mimic  the architecture of native tissues including reinforced nanofibers, woven scaffolds, non-woven scaffolds and microfibers. The typical manufacturing technique used is electrospinning which is advantageous because it creates a more accurate structural mimic of the native tissue extracellular matrix. 

In the study published by researchers at University of Utrecht Medical Center, use melt electrospinning. This electrospinning assembles the fibers layer by layer, supplying regulated control over assembly architecture. The researchers aimed to create a support for gelatin methacrylamide hydrogels with high porosity fiber scaffolds made of poly(ε-caprolactone) (PCL). The composite was created by infusing and crosslinking methacrylamide hydrogels within the PCL scaffolds. To stimulate the level of hydrogel reinforcement, a mathematical model was developed using the scaffold parameters. 

The study showed that the reinforced hydrogel stiffness was identical to that of articular cartilage as it increased up to 54-fold compared to hydrogels or microfiber scaffolds alone. The microfiber network can be used by various types of hydrogels which indicates that  they can offer mechanically and biologically favorable environments for various types of engineered tissues.

This current development in the field of tissue engineering will allow for the creation and use of resistant and effectual hydrogels to treat tissue loss or damage. The organized fibrous PCL scaffolds within the hydrogel allow for a healthy and diversified cell culture environment, because the hydrogel degrades over a few months which allows for new tissue to integrate into the scaffold. The PCL scaffold will in turn disintegrate within years, acting as a reinforcing network that will develop functional tissue. This reinforced hydrogel represents a step towards creating biomechanical functional tissue constructs and hopefully, more research will someday lead to the creation of the ideal, modify according to individual specifications engineered tissue replacement.



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3D-printed organ research enhanced with programmable DNA “smart glue”

Reporter: Irina Robu, PhD
A new breakthrough using DNA to provide the “glue” in a 3D printing material   was created by Dr. Andrew D. Ellington at University of Texas, which can be used to 3D print tissues to repair injuries or even create organs. Since DNA provides the source code for life, the researchers at University of Texas coated plastic microparticles with  40 base pairs of DNA, forming gel-like materials that they could extrude from a 3D printer* to form solid shapes (up to centimeters in size). These were used as scaffolds to host growing cells within the matrix.
The researchers designed DNA sequences which are complementary to one another and which are subsequently fragmented and attached to micro-beads and then anneal to one another and stiffen into a gel-like colloidal structure. The DNA-coated beads could carry and physically orient molecules that do more than simple annealing. The beads can also be coated with an increasing density of a growth factor leading a cell type to grow along the concentration gradient. 
In theory, this could lead to a future in which your outgoing organ is scanned and then its used to  design the large-scale structures of the printing scaffold. The small-scale structure of the tissues will be roughly the same for all livers, and the combination of beads necessary to create it sits ready in the laboratory freezer. Feed the scan and the right beads to the printer, along with samples of liver cells to be deposited in the gel as it is laid down. Print. Wait. Surgery.In addition to the benefits, there are also potential problems since DNA is fragile and the DNA interactions might not last enough for the organ to mature or the slow forming chemical bonds could lock a configuration in place once the DNA had figured it out transiently.As researchers customize XNAs to better offset DNA’s less helpful attributes, the potential will continue to grow.

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Superresolution Microscopy

Larry H. Bernstein, MD, FCAP, Curator


Lens-Free 3-D Microscope Sharp Enough for Pathology

LOS ANGELES, Dec. 18, 2014 —


A computational lens-free, holographic on-chip microscope could provide a faster and cheaper means of diagnosing cancer and other diseases at the cellular level.

Developed by researchers at the University of California, Los Angeles, the compact system illuminates tissue or blood samples with a laser or LED, while a sensor records the pattern of shadows created by the sample.

The device processes these patterns as a series of holograms using the transport-of-intensity equation, multiheight iterative phase retrieval and rotational field transformations. Computer algorithms correct for imaging artifacts and enhance contrast in the reconstructed 3-D images, which can be focused to any depth within the field of view even after image capture.
A tissue sample image created by a new chip-based, lens-free microscope. Images courtesy of Aydogan Ozcan/UCLA.
With a field of view several hundred times larger than that of an optical microscope, the lens-free device could considerably speed up diagnostic imaging. It is also much smaller than conventional microscopes.

“While mobile health care has expanded rapidly with the growth of consumer electronics — cellphones in particular — pathology is still, by and large, constrained to advanced clinical laboratory settings,” said professor Dr. Aydogan Ozcan. “Accompanied by advances in its graphical user interface, this platform could scale up for use in clinical, biomedical, scientific, educational and citizen-science applications, among others.”

The researchers tested the device using Pap smears that indicated cervical cancer, tissue specimens containing cancerous breast cells and blood samples containing sickle cell anemia.

In a blind test, a board-certified pathologist analyzed sets of specimen images created by the lens-free technology and by conventional microscopes. The pathologist’s diagnoses using the lens-free microscopic images were accurate 99 percent of the time, the researchers said.

“By providing high-resolution images of large-area pathology samples with 3-D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings,” the researchers wrote in Science Translational Medicine (doi: 10.1126/scitranslmed.3009850).

Funding for the project came from the Presidential Early Career Award for Scientists and Engineers, the National Science Foundation, the National Institutes of Health, the U.S. Army Research Office, the Office of Naval Research and the Howard Hughes Medical Institute.

Smartphone DNA measurements

Ozcan’s lab also recently demonstrated an optomechanical attachment that enables smartphones to perform fluorescence microscopy measurements on DNA.

The 3-D-printed device augments the phone’s camera by creating a high-contrast darkfield imaging setup with an inexpensive external lens, thin-film interference filters, a miniature dovetail stage and a laser diode for oblique excitation of fluorescent labels. The molecules are labeled and stretched on disposable chips that fit into the smartphone attachment.
A smartphone add-on enables imaging of DNA in the field.
The device also includes an app that transmits data to a server at UCLA, which measures the lengths of the individual DNA molecules.

This project was funded by the National Science Foundation. The results were published in ACS Nano (doi: 10.1021/nn505821y).

For more infromation, visit www.ucla.edu.

Expanded Tissues Show Confocal Microscopes More Detail

CAMBRIDGE, Mass., Jan. 26, 2015 —


Nobel Prize-winning superresolution microscopy techniques circumvent the diffraction limit of light to image the smallest details of cells. But there is another way.

Researchers at MIT have developed a method for making biological tissue samples physically larger, rendering their nanoscale features visible to conventional confocal microscopes.

Using inexpensive, commercially available chemicals and microscopes commonly found in research labs, the technique could give more scientists access to 3-D superresolution imaging.

“Instead of acquiring a new microscope to take images with nanoscale resolution, you can take the images on a regular microscope,” said MIT professor Dr. Edward Boyden. “You physically make the sample bigger, rather than trying to magnify the rays of light that are emitted by the sample.”

The process involves meshes of sodium polyacrylate, the superabsorbent chemical used in disposable diapers. When exposed to water, these meshes expand, and the cellular structures around them expand too.

Cells in rodent brain slices, as well as cells grown in vitro, are first fixed in formaldehyde and then gently stripped of their fatty membranes before being labeled with fluorescent markers.

A precursor is then added and heated to form the polyacrylate gel. Proteins that hold the specimen together are digested, allowing it to expand uniformly. Finally, the sample is washed in salt-free water to trigger the expansion.

Even though the proteins have been broken apart, the original location of each fluorescent label stays the same relative to the overall structure of the tissue because it is anchored to the polyacrylate gel by antibodies.
Scientists modified the superabsorbant diaper compound sodium polyacrylate to enlarge brain tissue and image it in 3-D using fluorescent tags and confocal microscopes. Courtesy of the Boyden Lab/MIT.
Samples were imaged before expansion using superresolution microscopes, and imaged afterward using confocal microscopes. Expansion gave the confocal microscopes an effective 70-nm lateral resolution — sharp enough to resolve details of the cell protein complexes, the spaces between rows of skeletal microtubule filaments and the two sides of synapses.


The diffraction limit means standard microscopes can’t resolve objects smaller than about 250 nm. “Unfortunately, in biology that’s right where things get interesting,” Boyden said.

Three inventors of superresolution microscopy won the 2014 Nobel Prize in chemistry. Superresolution techniques, however, have their own limitation: They work best with small, thin samples, and take a long time to image large samples. They can also be hampered by optical scattering in thick samples.

“If you want to map the brain, or understand how cancer cells are organized in a metastasizing tumor, or how immune cells are configured in an autoimmune attack, you have to look at a large piece of tissue with nanoscale precision,” Boyden said.

The MIT technique allowed imaging of samples approximately 500 × 200 × 100 µm in volume.

“The other methods currently have better resolution, but are harder to use, or slower,” said graduate student Paul Tillberg. “The benefits of our method are the ease of use and, more importantly, compatibility with large volumes, which is challenging with existing technologies.”

Funding came from the National Institutes of Health, National Science Foundation, New York Stem Cell Foundation, Jeremy and Joyce Wertheimer and the Fannie and John Hertz Foundation.

The research was published in Science (doi: 10.1126/science.1260088).

For more information, visit www.mit.edu.

Microscope Takes 3-D Images From Inside Moving Subjects

NEW YORK, Jan. 19, 2015 —


A new kind of microscope enables rapid 3-D imaging of living and moving samples, potentially offering advantages over laser-scanning confocal, two-photon and light-sheet microscopy.

Developed by Columbia University professor Dr. Elizabeth Hillman and graduate student Matthew Bouchard, swept confocally aligned planar excitation (SCAPE) microscopy involves simplified equipment and does not require sample mounting or translation. The microscope scans a sheet of light through the sample, making it unnecessary to position the sample or the microscope’s single objective.

“The ability to perform real-time, 3-D imaging at cellular resolution in behaving organisms is a new frontier for biomedical and neuroscience research,” Hillman said. “With SCAPE, we can now image complex, living things, such as neurons firing in the rodent brain, crawling fruit fly larvae and single cells in the zebrafish heart while the heart is actually beating spontaneously.”
SCAPE yields data equivalent to conventional light-sheet microscopy, but using a single, stationary objective lens; no sample translation; and high-speed 3-D imaging. This unique configuration permitted volumetric imaging of cortical dendrites in the awake, behaving mouse brain. Courtesy of Elizabeth Hillman/Columbia Engineering.
Conventional light-sheet microscopes use two orthogonal objectives and require that samples be in a fixed position. Confocal and two-photon microscopes can image a single plane within a living sample, but cannot generate 3-D images quickly enough to capture events like neurons firing.

SCAPE does have one drawback: Using a 488-nm laser, it cannot penetrate tissue as deeply as two-photon microscopy.

The new technique could be combined with optogenetics and other tissue manipulations, the researchers said. It could also be used for imaging cellular replication, function and motion in intact tissues, 3-D cell cultures and engineered tissue constructs; as well as imaging 3-D dynamics in microfluidics and flow-cell cytometry systems.

Hillman next plans to explore clinical applications of SCAPE, such as video-rate 3-D microendoscopy and intrasurgical imaging.

Funding for the project came from the National Institutes of Health, Human Frontier Science Program, Wallace H. Coulter Foundation, Dana Foundation and the U.S. Department of Defense.

The research was published in Nature Photonics (doi:10.1038/nphoton.2014.323).

For more information, visit www.engineering.columbia.edu.

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Rat Hearts Healed by a Protein-rich Gel

Reporter: Irina Robu, PhD

John Hopkins researchers  created a sticky protein rich gel which appear to help stem cells stay on or in rat hearts and have the ability to restore metabolism after transplantation in addition to improving cardiac function after simulated heart attacks.  When the heart beats, it pushes cells injected into the heart wall out in the lungs before they get a chance to attach to the wall.  John Hopkins researchers applied a hydrogel to the beating rat hearts to improve cell stem uptake to the heart muscle and speed up tissue healing after the heart attack.

In an effort solve the difficulties, M. Roselle Abraham, M.D. along with  Angel Chan, M.D., Ph.D. and  Jennifer Elisseeff, Ph.D. developed a hydrogel that combines serum, a protein-filled component of blood that contains everything cells need to survive, with hyaluronic acid, a molecule already present in the heart and in the matrix that surrounds and supports cells.

By mixing these two components, the researchers created a sticky gel that functioned as a synthetic stem cell niche: It encapsulated stem cells while nurturing them and rapidly restored their metabolism.

Their tests showed that encapsulated stem embryonic and adult stem cells survived at levels near 100 percent but still proliferated and survived for days.  According to their article being published in December 2015 issue of Biomaterials, when cell-gel combination was injected into the living hearts about 73% of cells were retained in the hearts after an hour and for the seven days the cells encapsulated into the hydrogel increased in number.

In rat models of heart attack damage, Abraham’s team shows that the hydrogel with encapsulated cells improved pumping efficiency of the left ventricle over the four weeks after injection by 15 percent, compared with 8 percent from cells in solution.  Abraham’s group showed that even injections of the hydrogel by itself improved heart function and increased the number of blood vessels in the region of the heart attack.



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