이 작품은 국립 보건원 (DBC에 HL068915), (CAP로) 채찍질 연구 기금에서 새 연구원 수상에서 연구 보조금, 보조금의 에이드 (DBC에 12GRNT11910008) 미국 심장 협회 (American Heart Association)에서보기에 의해 지원되었다 , 어린이 심장 재단 (DBC에), 그리고 보스턴 아동 병원 심장 전도 기금, 라이언 가족 성기 사이즈, 그리고 데이빗 풀먼에 의해 기부에서 연구 보조금.

여기에서 우리는 어린 양의 피부에서 COL1의 분리를 보여줍니다. 서면으로,이 프로토콜은 또한 성공적으로 토끼와 인간의 피부로부터 COL1를 분리하는데 사용될 수있다. 1. 피부 샘플을 준비 <ol><li> 사용하기 전에 4 ° C에 모든 시약을 평형. </li><li> 얼음처럼 차가운 dH보다 2 O의 피부 샘플 (25~50g)을 씻어 제모 크림 어떤 울, 모피, 또는 머리를 제거합니다. </li><li> 결합 조직과 지방의 샘플 깨끗하게 긁어 단일 에지 면도날을 사용합니다. </li><li> 얼음처럼 차가운 dH보다 2 O.에서 샘플을 씻어 </li><li> 단일 에지 면도날 1cm X 1cm 조각으로 피부 샘플을 조각. </li></ol> 2. Noncollagenous 가용화 물질을 제거 <ol><li> 샘플 50 ㎖ 당 원뿔 튜브의 5g을 달아 얼음처럼 차가운 0.5 M 아세트산 나트륨 30 ㎖를 추가합니다. </li><li> 벤치 탑 균질의 50 ML 튜브 어댑터를 사용하여 6m / 초 설정으로 1 분 동안 튜브를 섞는다. </li><li> SUP 폐기7 아세트산 나트륨 세척주기의 총 ernatant를 반복합니다. </li><li> 얼음처럼 차가운 dH보다 2 O에서 샘플을 씻어 잔류 아세트산 나트륨을 제거하기 위해 한 번 섞는다. </li><li> 물기를 제거하고 신선한 50 ML 원뿔 튜브로 전송 튜브의 측면에 대한 샘플을 압축하기 위해 주걱을 사용합니다. </li></ol> 3. I 콜라겐 추출을 입력 <ol><li> 샘플을 압축하고 각 세척 후 상층 액을 버리고 벤치 탑 균질 6 M / 초에서 1 분에 대한 ㎖ / g의 2 배의 샘플을 씻어 0.075 M 시트르산 나트륨 완충액. </li><li> 샘플에 0.075 M 시트르산 나트륨 완충액의 새로운 2 ㎖ / g 나누어지는을 추가합니다. </li><li> 각주기 사이의 버퍼를 제거하지 않고 6 연속 1 분, 교반 6m / 초 벤치 탑 균질 혼합주기를 수행합니다. </li><li> 포집 관에 두꺼운, 상등액을 전송합니다. </li><li> 샘플 ㎖ / g (1 건) 0.075 M 시트르산 나트륨 완충액을 추가하고 마지막으로 벤치 탑 균질화 AGITA을 수행기의 순환. </li><li> 포집 관이 최종 뜨는을 추가합니다. </li><li> 4 ℃에서 10 분 동안 3,200 XG에서 포집 관을 원심 분리기 </li><li> 100,000 분자량의 톱 수 납부에 뜨는을 전송 원심 필터 장치를 잘라. </li><li> 4 ℃에서 30 분 동안 3,200 XG에 원심 분리기 </li><li> 4 ° C에서 깨끗한 원뿔 관 및 저장에 상부 구획으로부터 정제 된 COL1 이동 </li></ol>

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저자는 공개 할 경쟁 금전적 이해 관계가 없습니다.

이 방법의 중요한 구성 요소는 물기를 제거하는 피부 샘플의 반복 효율적인 압축입니다. 이 샘플을 압축하여 단계 2.5에 극력 아세트산 나트륨을 제거하는 것이 중요하다. 우리는 튜브의 측면에 대해 피부를 압축 주걱을 사용하지만, 무명천도 사용될 수, 이것은 튜브에서 샘플의 제거를 필요로하고이 단계를 수행하는 데 필요한 시간을 증가시킬 것이지만. 마찬가지로, 시트르산 나트륨의 양이 후?...

수십 년 동안, 연구자 및 상업 벤더 사용할 수 있습니다 조직 COL1 원 섬유의 매트릭스의 재 부유 결과 중화 다음에 간단한 산 추출 프로토콜의 일부 변화를 이용하여 피부와 힘줄 등의 조직 소스의 구색에서 용해 COL1을 격리했다 생물 의학 응용 프로그램 1-4의 다수를위한. COL1에 대한 임상 응용의 많은 사례가 있지만이 단백질의 준비 5-7을 수행하는 일 또는 주를 복용 긴 추출을 필요로하기 때문에, 이들 중 몇몇은 autologously 파생 COL1를 사용합니다. 그 결과, 연구 조사와 의사는 일반적으로 쥐, 소 또는 돼지의 진피와 같은 인간이 아닌 조직을 사용하거나 시체에서 제거 스킨을 사용하거나 남성 할례에 따라 제조하는 COL1의 비용, 상업 준비를 사용합니다. 재생 응용 프로그램의 경우, 자신의 능력에 대한 이러한 제품의 전시 변화의 대부분은 고체 형성매트릭스 또는 세포 부착 및 성장을 지원할 수있는 조직 구조. 따라서, 신속하게 접근 할 수 있고 충분한자가 조직 소스에서 COL1를 추출하기 위해 디자인 된 새로운 표준화 된 방법에 대한 구별이 필요하다. 이러한 방법은 COL1은 또한 동물 모델에서 추출 콜라겐 계 지지체에 조립 설계 조직의 전임상 시험에 사용될 수 연구 설정에 시간과 비용을 절약한다. 동시에, 병원에서 급속 절연 autologously 유래 COL1를 사용하는 옵션을 갖는, 그렇지 않으면 동종 또는 이종 제제를받을 환자의 전체적인 안정성을 증가시킬 것이다. autogeneic 제제를 투여받은 환자의 경우,이 간소화 된 방법은 크게 생검 수집 및 콜라겐 애플리케이션 간의 간격을 줄일 것이다. 이러한 이유로, 우리는 고속 AG를 사용하여 노포 COL1 분리 및 정제 방법을 개선하고자itation 및 크기 배제 원심 분리. 이 방법은 많은 실험실에서 발견 된 표준 버퍼 및 장비를 사용하여 수행하는 것이 간단합니다. 고속 교반을 사용함으로써, 우리는 프로토콜 미만 3 시간 (8)에 대략 10 일 ~ 용해, 경피 COL1을 분리하는 데 필요한 시간을 감소시킨다. 중요한 것은,이 방법은 쉽게 절차의 단일 세트 동안 환자에 사용 autologously 유래 COL1을 준비하기 위해 임상 환경에서 수행 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="544">
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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
		</tr>
	</tbody>
</table>

an improved method for the preparation of type i collagen from skin

용해 1 형 콜라겐 (COL1)는 세포 배양을위한 접착제 기판으로 회생 응용 프로그램을위한 세포 지지체로 광범위하게 사용됩니다. 임상 적으로,이 단백질 널리 성형 수술, 피부 주사, 골 이식 및 재건 수술에 사용됩니다. 이러한 절차에 대한 COL1의 소스는 염증과 거부에 대한 가능성뿐만 아니라 생체 이물 질병의 전송을 증가하는, 일반적으로 인간이 아닌 있습니다. 이러한 관점에서, 효율적이고 신속 autologously 유래 조직의 한정된 양에서 COL1을 정화하는 방법은 많은 문제점을 회피 할하지만, 표준 격리 프로토콜 긴이며 종종 콜라겐 조직의 대량을 요구한다. 여기서, 우리는 이하의 3 시간에 약 10 일에서이 단백질을 분리 및 정제하는 데 필요한 시간을 줄여 효율적인 COL1 추출 방법을 설명한다. 우리는 많은 수술 과정 중에 있기 때문에 가능 여부에 대한 우리의 조직​​ 소스로 진피를 선택했다. 티그의 방법은 진피의 적은 양의 고순도, 수용성 COL1을 얻기 위해 강력한 교반 및 원심 여과와 함께 기존의 추출 버퍼를 사용합니다. 간단히, 피부 조직 검사는 지방, 결합 조직, 그리고 머리를 제거한 후 얼음처럼 차가운 dH보다 2 O 철저히 세척. 피부 샘플은 0.5 M 아세트산 나트륨 및 고속 벤치 탑 균질화기를 사용하여 단백질 및 다당류 noncollagenous 스트리핑된다. 잔류 고형물로부터 콜라겐이어서 균질화기를 사용하여 0.075 M 시트르산 나트륨 완충액으로 추출 하였다. 이 추출물을 비교하거나 우수한 상용 제품에 대한 품질이나 기존의 프로 시저를 사용하여 얻은 결과의 COL1 준비를 얻을 수 100,000 MW 컷 - 오프 원심 필터를 사용하여 정제된다. 우리는이 방법은 연구와 임상 응용 프로그램의 다수에 대한 autologously 파생 COL1의 활용을 용이하게 기대하고 있습니다.

이 COL1 격리 프로토콜은 완료하는 데 약 2 시간 15 분을 필요로한다. 그림 1은이 절차의 주요 단계를 요약 한 개략도를 보여줍니다. 샘플 준비 및 고속 동요의 12 사이클을 수행하고 아세트산 나트륨 헹굼은 약 35 분 (도 1a-C)가 소요된다. 한 교반을 수행하고 dH보다 2 O와주기를 씻어은 약 5 분 (그림 1D 및 1E)가 소요된다. 구연산 나트륨을 추가 한 교반에 샘...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

An Improved Method for the Preparation of Type I Collagen From Skin

수용성 제 1 형 콜라겐 (COL1)의 분리에 대한 기존의 절차 때문에 긴 버퍼 배양 및 섬유의 힘든 resuspensions의 처음부터 끝까지 약 10 일이 필요합니다. 여기, 우리는보다 3 시간에 작은 피부 생검에서 COL1을 정화 할 수있는 방법을 설명합니다.

피부에서 타입 I 콜라겐의 제조를위한 개선 된 방법

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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22.7K 조회수.  Boston Children's Hospital.  수용성 제 1 형 콜라겐 (COL1)의 분리에 대한 기존의 절차 때문에 긴 버퍼 배양 및 섬유의 힘든 resuspensions의 처음부터 끝까지 약 10 일이 필요합니다. 여기, 우리는보다 3 시간에 작은 피부 생검에서 COL1을 정화 할 수있는 방법을 설명합니다. 

비디오: An Improved Method for the Preparation of Type I Collagen From Skin

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

3 차원의 인간 피부 모델을 재구성 : 도구는 일반 피부와 흑색증의 진행을 공부하기

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

연구

생체공학

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypic 콜라겐 I 분석 : 3 차원 컨텍스트에서 셀 행동을 평가하기 위해 가단 플랫폼

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

줄기 세포 - 로드된 키토산 Microspheres의 배달을 위해 콜라겐 히드로겔을 구축

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

나노 크기의 지질 Vesicles의 준비를 위해 지속적인 압력 제어 압출 방법

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

방법 혈액 - 뇌 장벽의 인간 세포 기반의 연락 모델의 준비를 위해 개선

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

변성 콜라겐 물가 이미징<em&gt; 생체 내</em&gt;와<em&gt; 예 생체 내</em&gt; 리테이너 타입 콜라겐 미메시스 펩티드의 사진 트리거 하이브리드를 통해

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

뼈 이식 앵커리지 평가하는 향상된 기계적 시험 방법

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

압축 된 콜라겐에 다진 조직 : 단일 무대 재건 복구를위한 세포 함유 Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...