Calcaneal Bone Biopsy Inconsistency between Specimens Obtained From Trephine and Fine Needle Biopsy Techniques.

Standardized methods for osteomyelitis (OM) diagnosis of the lower extremity have proven to be difficult. Preoperative probability of foot osteomyelitis necessitates a combination of clinical, laboratory, imaging evidence (i.e., X-ray, CT, MRI), and bone biopsy to guide diagnosis and treatment. In the recent past, the relative weight that clinicians give to these collections of data to advise potential surgical intervention has been challenged, particularly with histologic evaluation of bone biopsy-traditionally considered "gold standard" in OM diagnosis. This study seeks to further expand this dialogue by retrospectively comparing calcaneal bone biopsies performed by direct visualization trephine approach (performed by Surgeons) vs fine needle biopsy with fluoroscopy guidance (performed by Interventional Radiologists). Results obtained from 57 patients with suspected calcaneal osteomyelitis demonstrate that Trephine obtained samples are significantly more likely to produce histopathologic evidence of OM (p-value: .013), microbiologic evidence of OM (p-value: <.001) and have better histopathologic and microbiologic concordance (p-value: <.001) than calcaneal bone biopsies obtained from Fine Needle Biopsy with fluoroscopy guidance.

A review of genetic engineering biotechnologies for enhanced chronic wound healing.

Traditional methods for addressing chronic wounds focus on correcting dysfunction by controlling extracellular elements. This review highlights technologies that take a different approach - enhancing chronic wound healing by genetic modification to wound beds. Featured cutaneous transduction/transfection methods include viral modalities (ie adenoviruses, adeno-associated viruses, retroviruses and lentiviruses) and conventional non-viral modalities (ie naked DNA injections, microseeding, liposomal reagents, particle bombardment and electroporation). Also explored are emerging technologies, focusing on the exciting capabilities of wound diagnostics such as pyrosequencing as well as site-specific nuclease editing tools such as CRISPR-Cas9 used to both transiently and permanently genetically modify resident wound bed cells. Additionally, new non-viral transfection methods (ie conjugated nanoparticles, multi-electrode arrays, and microfabricated needles and nanowires) are discussed that can potentially facilitate more efficient and safe transgene delivery to skin but also represent significant advances broadly to tissue regeneration research.

CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection.

BACKGROUND: CRISPR-Cas9 genome editing and labeling has emerged as an important tool in biologic research, particularly in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas9 plasmids to target cells is typically done by non-viral methods (chemical, physical, and/or electrical), which are limited by low transfection efficiencies or with viral vectors, which are limited by safety and restricted volume size. In this work, a non-viral transfection technology, named lance array nanoinjection (LAN), utilizes a microfabricated silicon chip to physically and electrically deliver genetic material to large numbers of target cells. To demonstrate its utility, we used the CRISPR-Cas9 system to edit the genome of isogenic cells. Two variables related to the LAN process were tested which include the magnitude of current used during plasmid attraction to the silicon lance array (1.5, 4.5, or 6.0 mA) and the number of times cells were injected (one or three times). RESULTS: Results indicate that most successful genome editing occurred after injecting three times at a current control setting of 4.5 mA, reaching a median level of 93.77 % modification. Furthermore, we found that genome editing using LAN follows a non-linear injection-dose response, meaning samples injected three times had modification rates as high as nearly 12 times analogously treated single injected samples. CONCLUSIONS: These findings demonstrate the LAN's ability to deliver genetic material to cells and indicate that successful alteration of the genome is influenced by a serial injection method as well as the electrical current settings.

The effect of injection speed and serial injection on propidium iodide entry into cultured HeLa and primary neonatal fibroblast cells using lance array nanoinjection.

BACKGROUND: Although site-directed genetic engineering has greatly improved in recent years, particularly with the implementation of CRISPR-Cas9, the ability to deliver these molecular constructs to a wide variety of cell types without adverse reaction is still a challenge. One non-viral transfection method designed to address this challenge is a MEMS based biotechnology described previously as lance array nanoinjection (LAN). LAN delivery of molecular loads is based upon the combinational use of electrical manipulation of loads of interest and physical penetration of target cell membranes. This work explores an original procedural element to nanoinjection by investigating the effects of the speed of injection and also the ability to serially inject the same sample. RESULTS: Initial LAN experimentation demonstrated that injecting at speeds of 0.08 mm/s resulted in 99.3 % of cultured HeLa 229 cells remaining adherent to the glass slide substrate used to stage the injection process. These results were then utilized to examine whether or not target cells could be injected multiple times (1, 2, and 3 times) since the injection process was not pulling the cells off of the glass slide. Using two different current control settings (1.5 and 3.0 mA) and two different cell types (HeLa 229 cells and primary neonatal fibroblasts [BJ(ATCC(®) CRL-2522™)], treatment samples were injected with propidium iodide (PI), a cell membrane impermeable nucleic acid dye, to assess the degree of molecular load delivery. Results from the serial injection work indicate that HeLa cells treated with 3.0 mA and injected twice (×2) had the greatest mean PI uptake of 60.47 % and that neonatal fibroblasts treated with the same protocol reached mean PI uptake rates of 20.97 %. CONCLUSIONS: Both experimental findings are particularly useful because it shows that greater molecular modification rates can be achieved by multiple, serial injections via a slower injection process.