Imagine you are Martin Birchall, laryngologist and ENT surgeon, star of regenerative medicine at UCL and trachea transplant enthusiast. You and your business partner Videregen need to explain to EU bureaucrats why your technology of decellurised cadaveric trachea is perfectly safe, what with all the dead patients of yours and your former best friend Paolo Macchiarini. Preclinical animal tests? Good idea indeed, though you have already published some very shady pig studies with Macchiarini in 2010, after you operated your first human patient in 2008 (whom you keep parading as success story of your stem cell magic superpowers, despite heavy complications which almost killed your research subject).

To distance yourself from that horrid Macchiarini, you do new preclinical tests. Enter three more pigs, which you decide transplant with a cadaveric decellurised human trachea. One trachea turns out to have been dirty not just with human “stem” cells, but also with pathogenic bacteria. So that one piggy got lucky and was set free. The unlucky other two: dead “of respiratory compromise”, one already after 12 days, the other suffered for a whole month after trachea transplant. You now have 100% preclinical mortality rate, and you still manage to convince the bureaucrats that the method works and 48 human patients must experience same, in the EU-funded phase 2 clinical trial TETRA. Thing is, that trachea transplant trial was supposed to help tracheal stenosis patients, by ridding them of burdensome stents and bronchoscopies, and not to euthanise them.

 

oink oink

Revealing EMA document

Unlike the two human patients of Birchall, who died because the stent inside their tracheal graft was removed or not inserted in the first place, the pigs had the advantage of the stent. It didn’t help them, though they died allegedly not of trachea transplant, but of living an unclean life in a pig sty.  Of course Birchall did not publish this dead pig story as a research paper. Nobody outside a closed circle knew about it, in fact this might have been one of the reasons the EU Commission fought tooth and nail to deny me access to TETRA documents. I got it following my FOIA inquiry from European Medicines Agency (EMA), and the full document “Application for orphan medicinal product designation” is here.

Large parts of the document, written by the trial sponsor Videregen, is blackened out. Here is the part about pigs, highlights mine:

“Two preclinical studies have been completed in the pig; the first study examined the surgical implantation of the MSC [mesenchymal stem cells from bone marrow (BM), can do magic,- LS] seeded scaffold transplanted at an orthotopic location with a four week follow up, and the second study examined the surgical implantation of the MSCs seeded scaffold transplanted at a heterotopic location supported by the administration of a Glyaderm® sheet [dermal replacement, made from human donor skin, -LS], in this instance preseeded with epithelial cells.
The choice of animal model for tissue engineering studies is challenging. Small animal models are not suitable as there are clear differences in tracheal size between rodents and humans. The pig model was chosen as the tracheal diameters, wall thickness and the ratio between the implant and the native trachea mimic the clinical situation and importantly the pig allowed the proposed clinical surgical procedures to be performed. However, the transplantation of the clinical product into the pig is a xenogenic situation and required immunosuppression to prevent graft rejection.
The animals, although housed in clean facilities, are not within an aseptic environment and the trachea transplanted animals are therefore subject to a range of airborne microbes with an increased risk of infection due to the immunosuppression. While anatomically similar to humans, the range of motion of the pig head and neck, the resultant biomechanical forces exerted on the trachea because of this and the quadrupedic nature of the animal, all have unknown effects on the outcomes of the implantation procedure and translation to the human clinical experience.

The first study was conducted to assess the feasibility, safety and efficacy of the first step of transplantation of a vacuum assisted DEM decellularised human trachea seeded with human BM derived MSCs in an orthotopic location [=in place of native trachea, -LS] in young adult domestic pigs. Three female pigs were assigned to the study and two underwent surgery. One tracheal scaffold was not released due to bacterial contamination prior to surgery and therefore the third animal was removed from the study.
After inducing general anaesthesia, an anterior midline cervical incision from the base of the larynx (cricoid) to the manubrium sterni was made to expose the trachea. The tracheal scaffold was trimmed according to the length of the stent to be implanted. A comparable length of native trachea was resected and the tracheal scaffold was inserted into the implantation area and the anastomoses were made. An appropriately sized stent was positioned and deployed.
The first animal was to be recovered for 36 days; however it died spontaneously at 32 days post surgery, without predisposing symptoms or condition. The second animal was to be recovered for 28 days, however it was terminated at 12 days post surgery due to signs of respiratory compromise incompatible with life. The study was stopped on humane grounds.
Post mortem examination revealed localised infection in both study animals. The infection was unlikely to have arisen from the seeded trachea as microbiology results at all points during manufacture were negative, including the final sample taken just prior to surgery. The one bacterial contaminated scaffold was identified before release and was not implanted. There was no evidence of surgery related changes in body weight or effects on haematology, clinical chemistry and coagulation parameters. IL 6 levels increased over the study period which could have been due to the bacterial infection of the tissue engineered tracheal scaffold”.

The next 2 paragraphs were blackened out, and even more remains black in the part describing an heterotopic transplantation of the trachea, i.e. not in place of the native trachea, but elsewhere in the pig’s body. It could have been subcutaneously, wrapped into muscle in a vain hope of re-vascularisation (which they themselves admit didn’t happen), or maybe shoved deep inside the pig’s anus: we cannot know as Birchall and Videregen made EMA censor that bit. Point remains: they didn’t try replacing a pig’s trachea again. Maybe ethic permission was denied? Birchall et al however did use a protective sheet of Gliaderm this time, as preservative for stem cells.

“It was clear from this study that minimising infection is required for successful tracheal transplantation A second study was conducted to assess the feasibility of administration of a Glyaderm® sheet seeded with epithelial cells into a MSC seeded trachea using porcine tracheal scaffolds. Four female pigs were assigned to this study.

[2.5 paragraphs blackened out]

Both surgical procedures were well tolerated. There was no evidence of intolerance of the tissue engineered tracheal scaffold or epithelial sheet based on body weight measurements, haematology, clinical chemistry and coagulation parameters. Clinical observations showed the animal remained clinically healthy throughout the study. Gross necropsy findings showed no visible infections or cysts, and very few adhesions. A fibrous capsule had formed around the tissue engineered tracheal scaffolds.
The explanted heterotopically located tissue engineered tracheal scaffolds showed very little sign of immune response, and the adventitial aspect showed signs of good vascularisation with numerous small vessels. On the luminal aspect, the tissue engineered epithelial sheet did not appear to be attached to the luminal surface of the tissue engineered tracheal scaffolds, and with only a few cells seen on the tissue engineered epithelial sheet facing the lumen. Vascularisation to the luminal surface of the tissue engineered trachea is required to support the implantation of an epithelial sheet.
In summary, the general surgical procedure of tracheal transplantation at both the orthotopic and heteroptopic location was tolerated in the pig. Small changes in clinical chemistry, haematology and coagulation were observed as would be expected following surgery.

Following the successful transplantation of tissue engineered trachea to an orthotopic location, infection led to early study termination. When the tracheal scaffold was placed at a heterotopic location with minimal risk of infections [which excludes pig’s anus after all! -LS], the scaffold did not become infected and retained good structural integrity. These data highlight the importance of minimising infection post tracheal transplant in a clinical setting. This can be achieved by placing an interim surface barrier (Glyaderm®) to minimise the risk of erosion of the tracheal surface due to infection”.

Vanishing pigs

Birchall has a knack of making pigs disappear. Another document I obtained under FOIA, this time from received from the UK Health Research Authority (HRA-NHS) is a set of documents concerning RegenVox, one of two phase 1 trachea transplant trials of Birchall’s suspended after my reporting (the other being Inspire, a trachea transplant trial on 4 patients). RegenVox is primarily about transplanting decellurised and “regenerated” larynx (voice box), but we learn from the original RegenVox grant application from April 2014 (starting page 12 here) that also the upper trachea was to be replaced in the same way, in up to 10 patients. That despite Birchall’s experience with the collapse of these tracheas, especially when defrosted grafts were used. In order to get RegenVox approved in the first place, Birchall claimed in the 2014 grant proposal to have done some pig experiments, on 8 animals:

“In a subsequent six-months’ survival study in 8 pigs, a two-step implantation procedure was used with improved anatomical reconstitution of the vocal cords. One died of an unrelated ear infection, but the remaining seven were healthy, eating and vocalising normal until euthanised. 3D CT scan reconstructions and histology demonstrated regeneration of cartilage and epithelium”

However, the follow-up paper reporting that data as Ansari et al, Stem Cell-Based Tissue-Engineered Laryngeal Replacement, Stem Cells Translational Medicine (2017), features only 6 pigs. Apparently, the other 2 pigs discovered an open sty window and flew away.

The UK Research Ethics Committee (REC) was concerned with the 20% porcine mortality rate (as reminder, this is about voice box transplant, not trachea, which is much more dangerous) and asked Birchall in June 2014 how he plans to prevent death incidents in human trial participants. Birchall explained :

“The mortality rate of 20% seen in the first pig study is not indicative of the expected mortality rate in this trial. There are important differences between this preclinical study and the RegenVOX trial. This preclinical study utilised a single stage implantation process and so the implants were not vascularised. Additionally, no epithelial sheet was used within the implants. The implants were therefore much more likely to fail due to poor vascularisation and infection. Using a 2 stage implantation process and lining the implant with an epithelial sheet is anticipated to reduce these risks”.

That was not entirely true. If one reads Birchall’s original RegenVox grant proposal, one can deduct from figure legends and his figure re-use that the 20% mortality did in fact apply to the Ansari et al 2017 paper with just 6 (never ever 8!) pigs proclaiming total safety of two-step decellurised larynx transplants.

 

Patricia Murray, professor in stem cell biology and regenerative medicine, who originally raised her concerns about Birchall’s human experimenting with the Science and Technology Committee of the British House of Commons, comments in this regard:

“It can thus be seen that the 81% survival rate reported for the first pig study mentioned in the REC submission and in the RegenVox proposal is from a study where the two-step procedure was used along with an epithelial sheet. It can therefore be concluded that false information was provided by the applicant in his response to this important question posed by the REC panel; i.e., the 20% mortality rate observed in the first pig study used the two-step procedure proposed in the clinical trial. Hence, the procedure described in RegenVox is not safe, and will likely lead to a 20% mortality rate in the human participants”.

A “field-test”

The most worrying part is not Birchall’s inability to tell the truth about his pig experiments, but about what happened to his human patients, as well as his attempts to twist that into a kind of success. The 19 year old Keziah Shorten, who received a decellurised cadaveric trachea from Macchiarini and his UCL partners, followed by a plastic trachea from Birchall, and then died because of both failed transplants, virtually ceased to exist. She is never mentioned in any of grant proposals of Birchall’s, also EMA was not informed of the gruesome outcome of that human experiment by UCL. Videregen’s “Application for orphan medicinal product designation” to EMA contains no mention of Keziah’s case whatsoever, not even in referenced papers.

The other young woman, 15 year old Shauna Davison (whose tragic story I previously told here and here) died just 2 weeks after the trachea transplant Birchall and his partner Martin Elliott imposed on her in early 2012. The tracheal collapse and death came so fast most likely because no stent was inserted, for whatever reason. Her death was much more difficult to hide, simply because Birchall and UCL repeatedly used her photograph for self-promotion, and even engaged a BBC documentary narrating her attempted rescue.

 

weiss shauna
Shauna used to advertise for UCL trachea transplants. From Weiss et al 2014: Tracheal bioengineering: the next steps. Proceeds of an International Society of Cell Therapy Pulmonary Cellular Therapy Signature Series Workshop, Paris, France, April 22, 2014

This is how Birchall et al described Shauna’s case in the RegenVox proposal (page 20):

“The third recipient (our second child patient) underwent a successful implant in 2011, but died of non-graft related causes soon afterwards. Her graft was healthy and had a good blood supply at the time of death. However and, importantly, her case afforded an opportunity to ‘field-test’ the Good Manufacturing Practice (GMP) procedures, protocols and quality control measures included in the present project”

The operation on Shauna was officially a compassionate use case, not a “field-test” of defrosted trachea without a stent. The claim that “her graft was healthy and had a good blood supply” was at the very best an educated guess of a UCL professor, there was no autopsy.  This is why in 2016 Videregen told a slightly different version to EMA (highlight mine):

“The last case was a 15 year old girl born with a single lung and major cardiac defect, with severe tracheal stenosis due to the scarring caused by recurrent stent re positioning and tracheotomy (Martin Birchall, personal communication). The narrowest part of the trachea was reduced to a few millimetres across. A multi disciplinary team reviewed suitability of the tracheal replacement, given her medical need for continuous care and risk of having had a cardiac arrest 4 months prior to surgery. The trachea was prepared similarly to that of the first case and allowed to mature in the bioreactor for 4 days.

Initial surgery was successful however the patient suffered what is assumed to be a fatal cardiovascular event 6 weeks following surgery during a bronchoscopy procedure to clear accumulating mucus that caused a tracheal obstruction and severe hypoxia. No post mortem was performed at the family’s request so cause of death cannot be confirmed.

Overall, therefore, there is a meaningful body of evidence to support the hypothesis that the tissue engineered product is a plausible approach to the treatment of tracheal stenosis.”.

Even the whitewash-investigation at UCL from 2017, which sole purpose was to save Birchall from himself (btw, did those investigators know about the two dead pigs?) determined that Shauna was suffocated by a collapsed tracheal graft which lacked a stent, only to recommend that more patients should be treated with same technology. Same investigation explained that Shauna was sent away from London to Leeds just days after her trachea replacement surgery because people were allegedly queuing to get same therapy and her hospital bed. It was in Leeds when Shauna’s tracheal graft collapsed and caused irreparable brain damage, two weeks after the operation. The girl died another week later, 3 weeks and not 6 after the experiment in London. Also unlike Videregen insinuated to EMA based on “personal communication” with Birchall, her death was certainly not the fault of Leeds doctors, who merely tried to save her life.

The trachea implanted into Shauna was frozen and defrosted, possibly on purpose to “field-test” the upscaling possibility freeze-storage offers to clinical trials. Birchall’s previous laboratory research showed that those defrosted grafts however are prone to collapse.  Also Keziah’s trachea and those of other cadaveric graft receipients of Macchiarini’s were stored frozen, as Macchiarini himself indirectly admitted in a lecture at thoracic surgery conference from May 2010, see minute 34:45 where he mentioned “frozen-kept airways”.

We do not know if the two unlucky pigs from the 2016 EMA application got defrosted or freshly prepared tracheas. In any case, both of them died quickly. Was the UK Medical Health products Regulatory Agency (MHRA), which eagerly greenlighted the phase 1 clinical trials Inspire and RegenVox, aware of those pigs? Did EU Commission bureaucrats read the EMA documents before deciding to give the phase 2 trial TETRA a chance? Finally, did UCL and their external investigators see that animal data when they announced that more animal tests would be unethical while calling for more human experimenting?


 

 

 

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